External ID: JHICC_RGS_Act_HTS

Protocol: Assay overview:

To screen for compounds that activate the RGS4 protein, a HEK293 cell line which stably expresses M3R and inducibly expresses RGS4 is employed. RGS4 function is monitored by calcium flux with a commercially available Fluo4-AM dye. Compounds that do not show increase in the Fluo4 fluorescence in induced RGS4 expressed cells are considered as activator/potentiator hits. M3 receptor and other endogenous receptor activators/potentiators will be excluded through later counter-screening against non-induced parental cells.

Protocol for RGS4 Primary Screen:
1. Cell culture: Cells (HEK293-FlpIn-TREx/M3R/RGS4) are routinely cultured in DMEM (high glucose, w/ glutamine), 10%FBS, 1%Pen/Strep, 15ug/ml Blasticidin, 400ug/ml G418, 200ug/ml Hygromycin.
2. Cell plating: Add 50 ul/well of 200,000 cells/ml re-suspended in DMEM/high glucose medium with 10% FBS, 1% Pen/Strep. Include 10 ng/ml Doxycyclin (DOX) to induce RGS4 expression.
3. Incubate overnight at 37C and 5% CO2.
4. Remove medium and add 20 ul /well of 2uM Fluo4-AM solution to cells.
5. Incubate 30 minutes at 37C in incubator.
6. Prepare 6x compound plates and control plates on Cybi-Well system: test compounds are prepared using assay buffer (HBSS-HEPES pH 7.4).
7. Remove Fluo4-AM dye solution and add 20 ul/well of assay buffer to cells.
8. Incubate 30 minutes at room temperature (RT).
9. Add 6x compounds in cell plates and incubate 20 minutes at RT.
9. Load cell plates on Hamamatsu FDSS 6000 kinetic imaging plate reader
10. Measure fluorescence for 10 seconds at 1Hz to establish baseline
11. After 100 seconds, add 4 ul of ECmax (carbachol) into the cell plates and record fluorescence for 100 seconds.
12. Calculate ratio readout as F(max-min)/F0 and integrated ratio readout.
13. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors [26]
14. Calculate B scores [27] for test compounds using integrated ratios calculated in Step 12
15. Outcome assignment: If the B score of the test compound is less than 4 times the standard deviation (SD) of the B scores of integrated ratios of all library compounds above the mean (B score Activator Ratio16. Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of Int(((Log(ABS(B score ratio))-0.82)/0.44)*100), and they are normalized to the smallest and largest LOG(B score ratio), B score ratio, as in the result definition. The inactive test compounds are assigned a score of 0.


List of reagents

1. HEK293-FlpIn-TREx/M3R/RGS4 cell lines (provided by assay provider)
2. PBS: pH7.4 (Invitrogen Cat#10010049)
3. Medium: DMEM (Sigma, Cat#D5796)
4. Fetal Bovine Serum (Gemini, Cat# 100-106)
5. Hygromycin (Mediatech, Cat#30-240-CR)
6. 100x Penicillin-Streptomycin (Mediatech, Cat#30-001-CI)
7. Cell/stripper (Mediatech, Cat#25-056-Cl)
8. G418: (Invitrogen, Cat#11811-031)
9. Blasticidin (Sigma, Cat#R21001)
10. Doxycycline hyclate (Sigma, Cat#D9891)
11. HEPES (Sigma, Cat#H4034)
12. Fluo-4 (Invitrogen, Cat #F14202)
13. Pluronic F-127*20% in DMSO (Invitrogen, Cat#P-3000MP)
14. Atropine (Sigma, Cat#A0132)
15. Carbachol (Sigma, Cat# C4382)
16. Triple-layer flask (VWR, Cat #62407-082)
17. BD Biocoat 384-well plates (BD, Cat# (35)4663 and Lot #7346273)

Comment: To screen for compounds that activate the RGS4 protein, a HEK293 cell line which stably expresses M3R and inducibly expresses RGS4 is employed. RGS4 function is monitored by calcium flux with a commercially available Fluo4-AM dye. Compounds that do not show increase in the Fluo4 fluorescence in induced RGS4 expressed cells are considered as activator/potentiator hits. M3 receptor and other endogenous receptor activators/potentiators will be excluded through later counter-screening against non-induced parental cells.

External ID: ADAM17_INH_QFRET_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that inhibit ADAM17. This assay employs a fluorophore and quencher pair. F =EDANS fluorophore, Q = DABCYL quencher. When intact, EDANS emission at 460nm is quenched by DABCYL via fluorescence resonance energy transfer. Upon cleavage of the scissile bond (A~V) by ADAM protease, the distance between fluorophore and quencher increases resulting in fluorescence increase at 460nm. Compounds are tested in singlicate at a final nominal concentration of 6.95 micromolar.

Protocol Summary:

Prior to the start of the assay, 2.5 microliters 2X ADAM17 enzyme (20 nM in Assay Buffer: 50 mM HEPES, 0.01% Brij, pH 7.5) are dispensed into 1536 microtiter plates. Compounds are added to plate (final concentration TBD) and incubated for 30 minutes at 25 degrees Celsius. The assay is started by dispensing 2.5 microliter of2X DM2 substrate (20 uM in Assay Buffer) to all wells. Plates are centrifuged and after 3 hours of incubation at 25 degrees Celsius, fluorescence is measured (excitation = 359nm, emission = 460nm).

The % inhibition for each well was then calculated as follows:

%_Inhibition = ( RFU_Test_Compound - MedianRFU_Low_Control ) / ( MedianRFU_High_Control - MedianRFU_Low_Control ) * 100

Where:

Test_Compound is defined as wells containing test compound.
High_Control is defined as wells treated with 1 micromolar Marimastat
Low_Control is defined as wells containing DMSO.


Interval Cutoff:
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Four values were calculated: (1) the average percent inhibition of all high controls tested plus three times the standard deviation of the high controls, (2) the average percent inhibition of all low controls tested minus three times the standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values, (3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity than the cutoff parameter was declared active.

PubChem Activity Outcome and Score:

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The activity score range for active compounds is 100-8, for inactive 8-0.

List of Reagents:

ADAM17 enzyme (R&D Systems, part # 930-ADB)
EDANS-DABCYL DM2 peptide substrate (Supplied by Assay Provider)
0.5M HEPES solution, pH7.5 (Teknova, part #101319-900)
Brij-35 (Sigma-Aldrich, part # P1254)
1536 well plate (Corning, part # 7261)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: BCCG-A405-UPR-XBP1-PrimaryAgonist-Assay

Protocol: UPR CHO-XBP1 1536-Well Assay Protocol
A. Brief Description of the Assay:
The purpose of this assay is to detect activators of the IRE1-XBP1 (adaptive) arm of the Unfolded Protein Response pathway.
B. Materials:
CHO-XBP1 Cell Line
F12 nutrient mix HAMs (Invitrogen, Cat# 11765047)
Fetal Bovine Serum, heat-inactivated (Hyclone, Cat# SH30396)
Penicillin/Streptomycin, liquid (Invitrogen, Cat# 15140122)
L-glutamine (100X ) (Invitrogen, Cat# 25030081)
MEM Non-Essential Amino Acids Solution 10 mM (100X) (Invitrogen, Cat# 11140050)
Trypsin-EDTA 0.25% (Invitrogen, Cat# 25200-056)
DPBS (Hyclone, Cat# 30028.02)
T225 TC Flask (Nunc, Cat# 159934)
Cell strainer, 40 um (BD, Cat# 352340)
Aurora 1536 well white solid bottom TC plate (Aurora Biotechnology 00029846)
Tunicamycin (Sigma-Aldrich, Cat# T7765)
Steady-Glo Luciferase Assay System (1L) (Promega, Cat# E2550)

C. Plate Map:
Positive control (P) in columns 1 and 2, 10ug/mL Tunicamycin
Negative control (N) in columns 3 and 4, No Tunicamycin
Test compound (T) in columns 5 - 48, No Tunicamycin

D. Procedures:
Day1 Cell Seeding
1. Prepare cell suspensions as described in section F. Cell culture.
2. Set up Kalypsys dispenser as described in section G. Kalypsys dispenser setting.
3. Plate 1000 cells/well in 5 uL of assay media into columns 1-48 of a 1536-well assay plate, using straight tip dispense on a Kalypsys dispenser.
4. Centrifuge plates at 500 rpm for 1 minute on Eppendorf centrifuge 5810. Use Kalypsys metal lids.
5. Incubate overnight at 37 degrees, 100% relative humidity, 5% CO2 for 16-18 hours.

Day2 Compound Addition
6. Using LabCyte Echo, transfer 50 nL from a 2 mM Echo qualified plate containing test compounds into assay plate Col. 5 - 48 (final concentration of test compounds is 20 uM, 1% DMSO), 50 nL of 1 mg/mL Tunicamycin in DMSO to positive control wells in Columns 1 and 2, and 50 nL of DMSO to negative control wells in Columns 3 and 4.
7. Centrifuge plates at 1000 rpm for 1 minute on a Vspin centrifuge.
8. Incubate plates in incubator (37 degrees, 100% relative humidity, 5% CO2) for 6 hours.
9. Set up Kalypsys dispenser and Perkin Elmer Envision as described in section G. Kalypsys dispenser setting and H. Envision setting.
10. Following 6 hours incubation, remove lid and incubate plate for 10 min in at room temp.
11. Add 3 uL of Steady-Glo to each wells (Columns 1 - 48) using a Kalypsys dispenser.
12. Centrifuge plates at 2000 rpm for 2 minutes on a Vspin centrifuge.
13. Lid plate and incubate for 10 min at room temp.
14. Read plates using Envision using a luminescence protocol.
E. Recipe:
Growth Media
F12 nutrient mix HAMs supplemented with 10% hi-FBS, 1X Penicillin/Streptomycin, 1X L-glutamine, and 1X MEM-NEAA
Assay Media
Filtered Growth Media
Trypsin
Dilute 0.25% Trypsin/EDTA to 0.05% Trypsin/EDTA using DPBS
Positive Control
Tunicamycin at 10 ug/mL final.
(Note: Tunicamycin is reconstituted in DMSO to a concentration of 10mg/ml).

F. Cell Culture:
Prepare Growth Media/Assay Media as described in section E. Recipe.
Procedure to expand and maintain cells:
CHO-CHOP cells are seeded into T225 flasks at 3.75 x 105 cells. Cells are passaged twice a week (Monday or Tuesday, and Thursday or Friday depending on cell growth). Confluency should be maintained at <75%. After 3 days incubation, ~2.5X10^7 cells are expected per T225 flask. Incubate cells in 5% CO2.
1. Put media in water bath and leave for 30 minutes. Also leave Trypsin at room temp for 15 minutes. Keep DPBS at room temp (no need to warm up DPBS at 37 degrees).
2. Aspirate off old media from T225 flask.
3. Wash the flasks with 20 mL DPBS per T225 flask. Leave cells in DPBS for about 30 seconds.
4. Add 6.5 mL 0.05% Trypsin solution into the flask. Rock the flask gently so that the solution covers all over the surface.
5. Allow the cells to detach by incubating at room temperature for about 4 minutes.
6. Wash the flask with 25 mL fresh growth media.
7. Collect the cell suspension in a 50 mL sterile conical tube.
8. Centrifuge at 900 rpm for 5 minutes. Once pelleted, remove the supernatant and resuspend the cell pellet carefully in 1 mL fresh growth media with p1000 pipette.
9. Add 19 mL of growth media and mix gently.
10. Filter the cell suspension with cell strainer.
11. Count the cells and prepare stock flasks with
3.00 x 10^5 cells per T225 flask for 4 days incubation.
3.75 x 10^5 cells per T225 flask for 3 days incubation.
Procedure to prepare cells for the assay:
Follow steps 1 - 3 above
4. Add 5 mL Trypsin into the flask. Rock the flask gently so that the solution covers all over the surface.
5. Allow the cells to detach by incubating at room temperature for about 4 minutes.
6. Wash the flask with 25 mL fresh growth media.
7. Collect the cell suspension in a 50 mL sterile conical tube.
8. Centrifuge at 500 rpm for 10 minutes. Once pelleted, remove the supernatant and resuspend the cell pellet carefully in 1 mL fresh assay with p1000 pipette.
9. Add 19 mL of assay media and mix gently.
10. Filter the cell suspension with cell strainer.
11. Count the cells and adjust cell density to 1.0x10^5 cells/mL in media.

Comment: Compounds that test with activity greater than or equal to 30% activation at 20 uM concentration relative to the positive control are defined as actives in the primary assay.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay demonstrated by a compound at 20 uM concentration:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay

External ID: JHICC_MrgX1_AlloAgonist_Primary

Protocol: Protocol:
1) Seed cells (MRGX1-HEK293) 15,000/50ul/well on BD PDK-pre-coated 384-well plate, overnight.
2) Dump the medium thoroughly and add 20 microl of Fluo4 NW solution.
3) 37 degrees C and 5% CO2 incubation in the dark for 0.5 hr and Room temperature incubation in the dark for 0.5 hr.
4) Mount the cell plate to FDSS, with compound plate from plate loading (1 by 1, 4 microl, 6x this step, 10x final); 2nd plate on Stage II (10 plate/2nd addition plate, 6 microl, 5x this step, 6.67x final) and 3rd plate on Stage III (10 plate/3rd addition plate, 6 microl, 6x this step, 6x final).
5) Read for the first 2 additions for 10 plates (~5 min/plate; total 55 min)
6) After that, read the 3rd addition of all 10 plates (~3 min/plate)
7) Change tips and repeat step 5 to step 7.
8) Calculate ratio readout as F(max-min)/F0 for the 2nd addition
9) Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors [6].
10) Calculate B scores [7] for test compounds using ratios calculated in Step 9.
11) Outcome assignment: If the B score of the test compound is more than 3 times the standard deviation (SD) of the B scores of ratios of the library compounds (>=3*SD), AND the B score of initial fluorescence intensity is within 5 times the standard deviation of the B scores of the library compounds, AND not as a MrgX1 agonist in 1st addition, the compound is designated in the Outcome as active as an allosteric agonist hit of the MrgX1 target. Otherwise, it is designated as inactive.
12) Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of with normalized ratioBScore, ratioBScore as in the result definition. Among the active compounds in the assay, the activity score range is 100-5. All inactive test compounds are assigned to the score 0.

List of reagents
(1) Cells (MrgX1 stably expressed HEK293 cells) from Dr. Xinzhong Dong.
(2) PBS: pH7.4 (Gibco, Cat#10010)
(3) Medium: DMEM (Mediatech 10-014-CV)
(4) Fetal Bovine Serum (Gibco, Cat# 26140)
(5) 200 mM L-Glutamine(Gibco, Cat#25030)
(6) Penicillin-Streptomycin(Mediatech, Cat#30-001-CI)
(7) Trypsin-EDTA: 0.25% Trypsin (Gibco, Cat#25300)
(8) G418 (100X): 50mg/mL(filtered) (Gibco, Cat#11811-031)
(9) HEPES (Sigma, Cat#H4034)
(10) BD Biocoat 384-well plates (BD, Cat#(35)4663 and Lot #7346273)
(11) Fluo-4 NW Calcium Assay Kit (Invitrogen Cat# F36205)
(12) BAM8-22 (Tocris, Cat#: 1763)

Comment: Possible artifacts of this assay can include, but are not limited to: non-intended chemicals or dust in or on wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, and compounds that quench or emit light or fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.

External ID: JHICC_MrgX1_Antagonist_Primary

Protocol: Protocol:
1) Seed cells (MRGX1-HEK293) 15,000/50ul/well on BD PDK-pre-coated 384-well plate, overnight.
2) Dump the medium thoroughly and add 20 microl of Fluo4 NW solution.
3) 37 degrees C and 5% CO2 incubation in the dark for 0.5 hr and Room temperature incubation in the dark for 0.5 hr.
4) Mount the cell plate to FDSS, with compound plate from plate loading (1 by 1, 4 microl, 6x this step, 10x final); 2nd plate on Stage II (10 plate/2nd addition plate, 6 microl, 5x this step, 6.67x final) and 3rd plate on Stage III (10 plate/3rd addition plate, 6 microl, 6x this step, 6x final).
5) Read for the first 2 additions for 10 plates (~5 min/plate; total 55 min)
6) After that, read the 3rd addition of all 10 plates (~3 min/plate)
7) Change tips and repeat step 5 to step 7.
8) Calculate ratio readout as F(max-min)/F0 for the 3rd addition
9) Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors [12].
10) Calculate B scores [13] for test compounds using ratios calculated in Step 8.
11) Outcome assignment: If the B score of the test compound is more than 3 times the standard deviation (SD) of the B scores of ratios of the library compounds (<=3*SD), AND the B score of initial fluorescence intensity is within 5 times the standard deviation of the B scores of the library compounds, AND not as a MrgX1 agonist in 1st addition, the compound is designated in the Outcome as active as an antagonist hit of the MrgX1 target. Otherwise, it is designated as inactive.
12) Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of with normalized ratioBScore, ratioBScore as in the result definition. Among the active compounds in the assay, the activity score range is 100-5. All inactive test compounds are assigned to the score 0.

List of reagents
(1) Cells (MrgX1 stably expressed HEK293 cells) from Dr. Xinzhong Dong.
(2) PBS: pH7.4 (Gibco, Cat#10010)
(3) Medium: DMEM (Mediatech 10-014-CV)
(4) Fetal Bovine Serum (Gibco, Cat# 26140)
(5) 200 mM L-Glutamine(Gibco, Cat#25030)
(6) Penicillin-Streptomycin(Mediatech, Cat#30-001-CI)
(7) Trypsin-EDTA: 0.25% Trypsin (Gibco, Cat#25300)
(8) G418 (100X): 50mg/mL(filtered) (Gibco, Cat#11811-031)
(9) HEPES (Sigma, Cat#H4034)
(10) BD Biocoat 384-well plates (BD, Cat#(35)4663 and Lot #7346273)
(11) Fluo-4 NW Calcium Assay Kit (Invitrogen Cat# F36205)
(12) BAM8-22 (Tocris, Cat#: 1763)

Comment: Possible artifacts of this assay can include, but are not limited to: non-intended chemicals or dust in or on wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, and compounds that quench or emit light or fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.

External ID: MH080376 Biochemical HTS for Inhibitors of the Proprotein Convertase Furin.

Protocol: Biochemical Furin HTS Assay protocol

Reaction Buffer Concentrations (final concentrations): 50 mM Hepes pH 7.5, 1 mM CaCl2, 1 mM beta-mercaptoethanol , 0.2 mg/ml BSA.

Substrate Stock Solution: 10 mM pERTKR-AMC in DMSO; stored in aliquots at -20 oC.

rhFurin714 prep (5.2 units/uL)Stored in aliquots at -80oC.

Furin Assay Protocol

The assay involves three liquid transfer steps of 5 uL each of 30 uM compound in 1% DMSO (10 uM final), 1 unit/5 uL rhFurin714 in 3X reaction buffer and 5 #L of 30 uM pERTKR-AMC substrate (10 uM final).

The assay plates used are Greiner 384-well, flat-bottom, low volume, black polystyrene plates (VWR catalog # 784076).

1. Add 5 uL of compound/controls to each well.
2. Add 5 uL of rhFurin714 in 3X buffer (1.0 units/well final).
3. Add 5 uL of 30 uM pERTKR-AMC fluorigenic substrate (10 uM/well final).
4. Incubate the plates for 1 hr at room temperature.
5. Stop the reaction by adding 5 uL of 1M Acetic acid.
6. Measure the amount of fluorigenic AMC released on the SpectraMax M5 using Ex = 345nm; Em = 440nm; cut-off 420 nm.

Comment: Active Criteria, Secondary Assay Plan, Hit and Lead Criteria.

Furin HTS Activity scoring rules:

The Furin inhibitor HTS run at the PMLSC utilized % inhibition calculated from maximum (n=32) and minimum (n=24) plate controls, with a hit criteria of >/= 25% inhibition to identify active compounds.

Furin Inhibitor scoring rules:

PUBCHEM_ACTIVITY_OUTCOME

1 - Substance is considered inactive when the % inhibition is < 25 %
2 - Substance is considered active when % activation is >/= 25 %
3 - Substance activity outcome is inconclusive

PUBCHEM_ACTIVITY_SCORE

0-40 scoring range is reserved for primary HTS data
a) if the % inhibition is >/= 25 %, the score is 40.
b) if the % inhibition is < 25 %, the score is 0.

Definition of Hit Criteria:
It is anticipated that all Furin inhibitor HTS actives will be confirmed in duplicate at the primary HTS concentration of 30 uM.
Furin inhibitor actives confirmed in the primary HTS format will then be run in 10-pt IC50 concentration response curves.
Confirmed hits will be subjected to structural confirmation by LCMS.

Secondary assay testing paradigm: Confirmed concentration dependent Furin inhibitors will be tested in the HeLa pcFur1.6 cell based ELISA assay.

External ID: Bursicon-induced LGR2 mediated cAMP production in LGR-2/CRE6x-Luciferase co-transfected HEK293 cells Inhibition - 7011-01_Antagonist_SinglePoint_HTS_Activity

Protocol: Protocol

Day 0:
Cell Culture
HEK 293 cells are plated in DMEM culture media (containing 10% Fetal Bovine Serum (FBS) and 1X penn/strep/glutamine) at a density of 5x106 cells/T175 flask and grown for 3 days at 37 degrees C in 5% CO2 incubator.

Day 3:
Transient Transfection
Cells are transiently transfected in DMEM transfection media (containing 1X penn/strep/glutamine without serum) using polyethyleneimine (PEI).
1)Mix 2.9ug/flask of cDNA encoding the wild-type Bursicon Receptor with 14.6ng/flask of cDNA encoding a CRE-luciferase reporter gene with a PEST/HCL sequence in 2mL of transfection media
2)Add 61uL/flask of PEI of 1mg/mL in 1.5mL of transfection media
3)Mix the 2mL cDNA solution with 1.5mL of PEI solution and incubate at RT for 20 mins
4)Aspirated the culture media from the cells in T175 flask
5)Add 25 mL of transfection media and 3.5mL of transfection mixture to T175 falsk.
6)Mix the media with transfection mixture well and transfect for 2 days at 37 degrees C in 5% CO2 incubator

Day 5:
Cell Plating
1)Typsinize the Cells with 5mL of 0.05% Trypsin and incubate at 37oC for 5 min.
2)Add 5mL of DMEM assay media (containing DMEM without phenol red, 10% NuSerum and 1x Pen/Strep/Glutamine) to inactive trypsin
3)Collect cells by centrifugation at 1000rpm for 5 min
4)Suspend the cells in DMEM assay media to a cell density of 4x105 cells/mL
5)Plate the cells to 1536-well Aurora Mako plate with 2000 cells/well/5uL using ViaFill
6)Incubate the cells at 37 degrees C in 5% CO2 incubator on GS system for overnight

Day 6:
Compound Treatment, Agonist Stimulation and Detection
Assay Setup
1)Thaw aliquoted Bursicon conditioned media and diluted with DMEM assay media at a 1/20 dilution. Filter the solution through a 0.22uM filter
2)Add DMEM condition media, the diluted Bursicon conditioned media and SteadyGlo to the bottles and setup BioRAPTR

Run automation protocol on GS system
1)Take the assay plate with transfected cells from the incubator
2)Transfer 5nL/well of 10mM compound to assay plate
3)Incubate 30 mins in incubator
4)Add 1uL/well of DMEM assay media to PosCon wells and diluted Bursicon solution to the other wells using BioRAPTR (Beckman) based on the plate map
5)Incubate the assay plate for 4 h in incubator
6)Take the plate out from the incubator and equilibrate the assay plate at RT for 10 mins
7)Add 1uL/well of SteadyGlo to assay plate using BioRAPTR, incubate at RT for 10 mins
8)Read the plate on ViewLux (PerkinElmer) for Luminescence.

Culture Media
DMEM high glucose, no glutamine (Invitrogen, 11960)
Pen/Strep/Glutamine (Invitrogen 10378-016)
Fetal Bovine Serum (Atlanta Biological, S10350)

Transfection Media
DMEM high glucose, no glutamine (Invitrogen, 11960)
Pen/Strep/Glutamine (Invitrogen 10378-016)

Assay Media
DMEM no phenol red (Invitrogen, 21063-029)
Pen/Strep/Glutamine (Invitrogen 10378-016)
NuSerum (BD biosciences, 24883)

0.05% Trypsin-EDTA (Invitrogen, 25300-062)
Aurora 1536-well Mako plate (Aurora/Brooks, 00028778)

Comment:

External ID: HMS1149-MLP

Protocol: A 558 bp region upstream of hspX was PCR amplified and cloned upstream of the GFP-mut2 (Cormack et al., 1996). This construct was cloned into a modified version of the replicating plasmid pSE100 and transformed into CDC1551 to generate CDC1551(hspX'::GFP).

The M. tuberculosis CDC 1551 (hspX'::GFP) fluorescent biosensor was grown in pH 7.0 Middlebrook 7H9 medium to mid- to late-log phase. Black-walled, clear-bottom, tissue-culture treated 384-well microtiter plates (Corning #3712) were filled with 25 uL of buffered pH 7.0 7H9 medium using a Matrix Combi. 100 nL of compound was then transferred to each well via stainless steel pin array. Plates were manually sealed with DMSO-resistant aluminum seals and frozen at -80C until they were ready to be screened. The cultures were re-suspended in pH 7.0 7H9 and 25 uL was dispensed into each well of the 384-well assay plates already containing the perturbator utilizing a Cy-Bio Selma liquid handler robot to an OD595 of 0.05. The plates were then placed in a Ziploc bag with a moistened paper towel (to limit evaporation) and incubated for 6 days at 37C. Fluorescence and optical density (OD) readings were made on an EnSpire plate reader (Perkin Elmer, Inc.) at excitation and emission wavelengths of 488 and 509 nm as a top read, with the OD being taken at 595 nm as a bottom read.

The ~328,633 compound library was arrayed into black-walled, clear-bottom tissue-culture treated 384-well microtiter plates (Corning #3712) by pin transfer (100 nL), containing 25 uL of buffered pH 7.0 7H9 medium. In a previous screen utilizing the same compound library and screening method, four random compound plates were screened in duplicate to observe variation in hit robustness across plates and method. The result was that the duplicate plates did not vary in a statistically significant manner and due to the high volume of compounds to be screened (~328,633), replicates were not utilized.

Negative control: DMSO, 0.1% final concentration in buffered pH 7.0 7H9 in column 2 or column 23 of each plate

Positive control: 0.3 uM Rifampicin final concentration in buffered pH 7.0 7H9 in column 1 or column 24 of each plate

Comment: Analysis methods:
To calculate normalized fluorescence percent inhibition, plate average negative control fluorescence intensity (based on all plates within a screening run) was subtracted from well fluorescence intensity, divided by the difference between plate negative and positive control average fluorescence intensity (based on all plates within a screening run), and multiplied by 100. To calculate normalized growth inhibition, plate average negative control absorbance (based on all plates within a screening run) was subtracted from well absorbance, divided by the difference between plate negative and positive control average absorbance (based on all plates within a screening run), and multiplied by 100. Normalized fluorescence percent inhibition was divided by normalized growth inhibition to calculate a ratio. Wells were considered active if growth was not greatly decreased and showed either a 3-fold selectivity fluorescence to growth inhibition ratio (with 35-45% fluorescence inhibition) or a 1.5-fold selectivity fluorescence to growth inhibition ratio (with > 45% fluorescence inhibition). These compounds may be directly or indirectly inhibiting DosRS signaling. Wells were considered general inhibitors of M. tuberculosis growth if both fluorescence and growth inhibition > 50%. Activity scores were scaled from 100 to 0 and derived from normalized fluorescence percent inhibition, with 100 (or > 100) = 100% inhibition and 0 (or < 0) = no inhibition.

External ID: ABHD4_INH_FP_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this biochemical assay is to identify compounds that act as inhibitors of alpha/beta hydrolase domain containing 4 (ABHD4). In this assay, ABHD4 protein is incubated with test compounds and fluorophosphonate-rhodamine (FP-Rh) activity-based probe. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value (mP). As designed, test compounds that act as ABHD4 inhibitors will prevent ABHD4-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds are tested in singlicate at a nominal test concentration of 8.92 micromolar.

Protocol Summary:

Prior to the start of the assay, 4 microliters of assay buffer (50 mM HEPES pH 7.0, 150 mM NaCl and 0.01% Pluronic F-127) were dispensed into column 2 only. Next, 4 microliters of assay buffer containing 46.9 ug/mL of ABHD4 mutant protein (S159A) were dispensed into columns 1 and 3 of 1536 microtiter plates and 4 microliters of assay buffer containing 46.9 ug/mL of ABHD4 wild type protein were dispensed into columns 4 thru 48. Then, 45 nL of test compound in DMSO or DMSO alone (0.89% final concentration) were added to the appropriate wells and incubated for 45 minutes at 25 degrees Celsius.

The assay was started by dispensing 1.0 microliter of 188 nM FP-Rh in assay buffer to all wells. Plates were centrifuged and after 60 minutes of incubation at 25 degrees Celsius, fluorescence polarization was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a BODIPY dichroic mirror (excitation = 525nm, emission = 598nm) for 30 seconds for each polarization plane (parallel and perpendicular).

Prior to further calculations, the following formula was used to calculate fluorescence polarization (FP):

FP = ( Raw1 - Raw2 ) / ( Raw1 + Raw2 )

Where:

Raw1 is defined as the S channel.
Raw2 is defined as the P channel.

The percent inhibition for each compound was calculated as follows:

100 *( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )

Where:

Test_Compound is defined as wells containing ABHD4 wild type protein in the presence of test compound and FP-Rh.
High_Control is defined as wells containing ABHD4 mutant protein, DMSO and FP-Rh.
Low_Control is defined as the median of the wells containing test compounds.

PubChem Activity Outcome and Score:


A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The activity score range for active compounds is 100-6, for inactive 6-0.

List of Reagents:

mABHD4 wild type protein (supplied by Assay Provider)
mABHD4 (S159A) mutant protein (supplied by Assay Provider).
FP-Rh probe (supplied by Assay Provider)
HEPES (Sigma, part 83264)
NaCl (Sigma, part S6546)
Pluronic F-127 (Invitrogen, part P6866)
1536-well plates (Corning, part 7261)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: HIV1-VIF_MS

Protocol: The assay protocol for screening and testing compounds included plating a mixture of GST-Vif "donor" protein and bio-A3G "acceptor" peptide with test compounds in white, non-binding surface 1536-well plates. LANCE reagents were purchased from PerkinElmer. Briefly, 15nM GST-Vif protein was added to the assay plate containing test compounds. Following a 30 minute incubation, 500 nM bio-A3G peptide was added. Following another 30 minute incubation, 2 nM Eu-labeled anti-GST and 50 nM streptavidin-Ulight detection reagents were added to the assay plate and incubated for 1 hour. After the final incubation, TR-FRET signals were measured using an Envision microplate reader with excitation at 340 nm and emission at 615 and 665 nm. The emission at 615 nm from Eu-labeled anti-GST induces emission at 665 nm from Ulight conjugated to streptavidin when the two molecules are in close proximity, resulting in a FRET signal. GST-Vif binding to the bio-A3G peptide produced a significant increase in the FRET signal over background levels measured in the presence of GST.

To evaluate the screening results each plate included negative and positive controls. Negative control wells included all assay components in the absence of an inhibitor and positive control wells included all assay components with GST-Vif 1-94 protein replaced by GST protein. Percent inhibition was determined as: 100*((Test Compound Signal - Median Negative Control Signal)/(Median Positive Control Signal - Median Negative Control Signal)).

Comment: In the Dose Response, compounds that showed >30% inhibition for at least one concentration were considered potentially active, however some compounds were tested in dose response on more than one occasion. If conflicting results were observed, the compound was deemed "inconclusive". Only compounds that consistently showed activity across all test dates were labeled "Active". Any compounds in the primary screen active range that were not available for testing in dose response were labeled "Inconslusive".
The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score. Compounds in the primary screen are scored on a scale of 0-40 based on inhibitory activity where a score of 40 corresponds to 100% inhibition. In the confirmatory dose response screen, active compounds were scored on a scale of 41-80 based on the average IC50 result, inconclusive compounds were given the lowest confirmatory score of 41, and compounds that were tested and did not confirm as actives were given the score 0.

External ID: SIAE_INH_FP_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this biochemical assay is to identify compounds that act as inhibitors of sialic acid acetylesterase (SIAE). In this assay, SIAE protein is incubated with test compounds and fluorophosphonate-rhodamine (FP-Rh) activity-based probe. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value (mP). As designed, test compounds that act as SIAE inhibitors will prevent SIAE-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds are tested in singlicate at a nominal test concentration of 9.66 micromolar.

Protocol Summary:

Prior to the start of the assay, 3 microliters of assay buffer (1X DPBS and 0.01% Pluronic F-127) were dispensed into column 1 thru column 3 of 1536 microtiter plates. Next, 3 microliters of assay buffer containing 0.73uM of SIAE protein were dispensed into columns 4 thru 48. Then, 39 nL of test compound in DMSO or DMSO alone (0.97% final concentration) were added to the appropriate wells and incubated for 45 minutes at 25 degrees Celsius.

The assay was started by dispensing 1.0 microliter of 300 nM FP-Rh in assay buffer to all wells. Plates were centrifuged and after 120 minutes of incubation at 25 degrees Celsius, fluorescence polarization was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a BODIPY dichroic mirror (excitation = 525nm, emission = 598nm) for 25 seconds for each polarization plane (parallel and perpendicular).

Prior to further calculations, the following formula was used to calculate fluorescence polarization (FP):

FP = ( Raw1 - Raw2 ) / ( Raw1 + Raw2 )

Where:

Raw1 is defined as the S channel.
Raw2 is defined as the P channel.

The percent inhibition for each compound was calculated as follows:

100 *( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )

Where:

Test_Compound is defined as wells containing SIAE protein in the presence of test compound and FP-Rh.
High_Control is defined as wells containing DMSO, FP-Rh but, no SIAE protein.
Low_Control is defined as the median of the wells containing test compounds.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Four values were calculated: (1) the average percent inhibition of all high controls tested plus three times the standard deviation of the high controls, (2) the average percent inhibition of all low controls tested minus three times the standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values, (3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity than the cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The activity score range for active compounds is 100-2, for inactive 2-0.

List of Reagents:

SIAE protein (supplied by Assay Provider)
FP-Rh probe (supplied by Assay Provider)
DPBS (Mediatech, part 20-031-CV)
Pluronic F-127 (Invitrogen, part P6866)
1536-well plates (Greiner, part 789176)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: 7052-01_Inhibitor_SinglePoint_HTS_Activity_Set2

Protocol: Caspase 6 enzyme was received from collaborator and stored at -80 C. Stock concentration was at 1.2 mg/ml (43.1 uM).

Assay Buffer:
100 mM Hepes pH 7.5, 10% sucrose, 0.1 % CHAPS, 5 mM DTT (final concentration) was added fresh just prior to assay

Reagents:
VEID-R110-VEID: Caspase 6 substrate, was reconstituted to 10 mM from dry powder with DMSO and stored at -20 C.
VEID-CHO: Caspase inhibitor (positive control) was reconstituted to 10 mM from dry powder with DMSO and stored at -20 C.

Assay Ready Plates:
1536 black, solid bottom plates containing 7.5 ul of compound

Protocol:
Add 4 ul 2.2 nM Caspase-6 to 1536 ARPS using BioRaptR
Add 100 nL VEID-CHO to positive control wells using Combi nL
Incubate Room temp 60 min
Add 2 ul of 24 uM VEID-R110-VEID (Caspase 6 substrate) to all wells
Incubate at room temp for 40 min
Read plate on Envision (FITC settings and filters)

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.


PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v10.0.2) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 35.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

External ID: PROCASPASE3_ACT_EPIABS_1536_1X%ACT PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that activate procaspase 3 activity. This assay employs a procaspase 3 mutant enzyme, D9A/D28A/D175A (called PC-3 D3A) which is unable to autoproteolyze itself because its aspartic acid cleavage sites have been mutated to alanines. The mutant has a fully functional active site that can process the peptidic Ac-DEVD-pNA chromogenic substrate. Cleavage of substrate by procaspase 3 hydrolyzes the bond between the aspartic acid and p-nitroaniline, leading to release of yellow p-nitroaniline and an increase in well absorbance at 405 nm. In this assay, PC-3 D3A enzyme is pre-incubated with test compounds, followed by addition of substrate and measurement of well epi-absorbance. As designed, compounds that activate procaspase 3 activity will increase substrate hydrolysis, leading to an increase in well absorbance. Compounds are tested in singlicate at a nominal concentration of 8.5 uM.

Protocol Summary:

Prior to the start of the assay, 2.5 uL of zinc-free Assay Buffer (50 mM HEPES, 300 mM NaCl, pH 7.4, 0.01% Triton-X 100) containing 2 uM of PC-3 D3A protein were dispensed into 1536 microtiter plates. Next, 43 nL of test compound in DMSO or DMSO alone (0.8% final concentration) were added to the appropriate wells and incubated for 1 hour at 25 C.

The assay was started by dispensing 2.5 uL of 400 uM Ac-DEVD-pNA in Assay Buffer to all wells. Plates were centrifuged and after 2 hours of incubation at 25 C, epi-absorbance was read on the EnVision plate reader using a photometric filter set (excitation = 405 nm, emission = 450 nm) and a dichroic mirror with 425 nm cutoff. Fluorescence emission was read at 10 flashes per well at two time points (0 minutes and 120 minutes).

Prior to further calculations, the following formula was used to calculate absorbance:

Abs = ( -Log10( ( [ Raw2 ] / [ Mean Reference2 ] ) ) - ( -Log10 ( ( [ Raw1 ] ) / [ Mean Reference ] ) )

Where:

Raw1 is defined as the read at T0 minutes.
Raw2 is defined as the read at T120 minutes.
Mean Reference is defined as a mean of values from wells containing buffer only at T0.
Mean Reference2 is defined as a mean of values from wells containing buffer only at T120.

The percent activation for each compound was calculated as follows:

% Activation = ( ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) ) * 100

Where:

Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing DMSO.
High_Control is defined as wells containing DMSO and 5 uM PC-3 D3A.

A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated: (1) the average percent activation of all compounds tested, and (2) three times their standard deviation.The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % activation than the cutoff parameter was declared active.

PubChem Activity Outcome and Score:

The reported PubChem Activity Score has been normalized to 100% observed primary activation. Negative % activation values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-9, and for inactive compounds 9-0.

List of Reagents:

Recombinant PC-3 D3A (procaspase 3 enzyme) (Assay Provider)
Assay Buffer (Assay Provider)
Chromogenic Substrate (Ac-DEVD-pNA) (Assay Provider)
1536 SWSN plates (Corning, part 7254)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well absorbance. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: UHRF1-CPGDNA_INH_TRFRET_1536_1X%INH CSRUN for MBD2-CPGDNA INH

Protocol: Assay Overview:

The purpose of this assay is to determine whether compounds identified as inhibitors of MBD2 are nonselective, as determined by inhibition of methylated DNA-binding activity of the SRA domain polypeptide of ubiquitin-like with PHD and ring finger domains 1 (UHRF1). The UHRF1 gene encodes a member of a subfamily of RING-finger type E3 ubiquitin ligases. It contains a Set-and RING Associated (SRA) domain that binds to partially methylated DNA sequences during DNA synthesis. UHRF1 exhibits a different mode of DNA binding than MBD2, binding so-called hemi-methylated cytosines in order to promote their fully methylation. This assay is performed in a similar manner as the MBD2 screen, except that it employs recombinant UHRF1 SRA domain protein (UHRF1_SRA) and hemi-methylated DNA as its substrate. This assay monitors the ability of compounds to inhibit binding of cloned recombinant UHRF1_SRA to a FAM-labeled 14 bp hairpin oligonucleotide containing a hemimethylated CpG. In this biochemical assay, test compounds are incubated with UHRF1_SRA and a 5-carboxyfluorescene (FAM)-labeled 14bp hairpin DNA oligonucleotide containing one hemimethylated CpG dinucleotide in the presence of Tb-labeled anti-His antibody. An inhibitor of the UHRF1_SRA:FAM hemimethylated CpG interaction will inhibit binding between UHRF1 and FAM hemimethylated oligo, leading to reduced energy transfer and reduced well FRET (fluorescein emission / Tb emission). Compounds are tested in singlicate at a final nominal concentration of 4.4 micromolar.

Protocol Summary:

The assay was started by dispensing 5 microliters of Control Mix in assay buffer (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 125 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 0.2% Tween-20) containing 125 nanomolar of UHRF1 protein, 5 micromolar of FAM labeled methylated CpG DNA oligo and 5 nanomolar of Terbium labeled anti-his antibody into columns 1 thru columns 2 of 1536 microtiter plates. Next, 5 microliters of Experimental Mix containing 125 nanomolar of UHRF1 protein, 100 nanomolar FAM labeled Hemimethylated CpG DNA oligo and 5 nanomolar Terbium labeled anti-his antibody in assay buffer were dispensed into columns 3 thru 48. Then, the plates were centrifuged and pinned with 22 nL of test compound in DMSO, Mitoxantrone (1.3 micromolar final concentration) in DMSO or DMSO alone (0.44% final concentration). The plates were incubated for 60 minutes at 25 degrees Celsius and TR-FRET was measured on a Viewlux microplate reader (Perkin Elmer, Turku, Finland). After excitation at 340 nm, well fluorescence was monitored at 495 nm (Tb) and 525 nm (FAM). For each well, a fluorescence ratio was calculated according to the following mathematical expression:
Ratio = I525nm / I495nm

Where:

I525nm represents the measured fluorescence emission at 525 nm.
I495nm represents the measured fluorescence emission at 495 nm.

The percent inhibition for each compound was calculated using as follows:

%_Inhibition = 100 * ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) )

Where:

Test_Compound is defined as wells containing the Experimental Mix in the presence of test compound.
High_Control is defined as wells containing the Experimental Mix, Mitoxantrone and DMSO.
Low_Control is defined as the median of the wells containing test compounds.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

The reported Pubchem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-19, and for inactive compounds 19-0.

List of Reagents:

UHRF1_SRA protein (supplied by Assay Provider)
FAM labeled Hemimethylated CpG DNA Oligo (IDT-custom order)
FAM labeled Methylated CpG DNA Oligo (IDT-custom order)
Lanthascreen Tb Anti-His Antibody (Invitrogen, part PV5895)
MgCl2 (Fisher, part BP214)
NaCl (Sigma, part S6546)
DTT (Fisher, part BP172)
EDTA (Sigma, part E7889)
Tris (Amresco, part 0497)
Tween 20 (Sigma, part P9416)
Glycerol (Sigma, part G7893)
1536-well plates (Corning, part 7261)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: SBCCG-A1016-RevErbaLBD-Primary-Assay

Protocol: This assay is to identify modulators of Rev-erb alpha protein binding to DNA

A. Materials:
REV-alpha_beta purified protein = Rastinejad lab
FITC-DNA = Rastinejad lab
Tris = Biorad (cat #161-0719)
DTT = Akron Biotechnology (cat #AK2948-0005)
5M NaCl = Sigma-Aldrich (cat #56546-1L)
Glycerol 99.5% = Acros (cat #327255000)
Tween 20 = Sigma-Aldrich (cat #P1379)
Corning high base black plates = Corning (cat #3724)
Molecular grade water = Cellgro (cat #46-000-CM)

B. Plate Map:
Negative (low) control in columns 1 and 2 is 6nM DNA and 35nM Protein, DMSO
Positive (high) control in columns 3 and 4, Protein at 750nM and 6nM DNA, DMSO
Test compound in columns 5 - 48, Protein at 35nM + 6nM DNA + test compound

C. Procedures:
Step#Description
1#Prepare 2X Rev-erb alpha protein stock and 2X DNA stock
2#Using LabCyte Echo, transfer xnL from a 2 mM Echo qualified plate containing test compounds into assay plate Col. 5 - 48. Add same volume of DMSO in col 1-4.
3#Spin plates at 1000 rpm for 1 minute in centrifuge.
4#Using the bioraptr, add 3 uL/well of (35nM protein control) to columns 1 and 2 and test compound wells.
5#Using the bioraptr, add 3 uL/well of Mix 2 (750nM protein) to col. 3-4 for the positive control
6#Using the bioraptr, add 3uL/well of Mix 3 (DNA) to col. 1-48.
7#Spin plates at 1000 rpm for 1 minute in centrifuge.
8#Incubate plates in the dark at room temperature for 90 minutes.
9#Set up Perkin Elmer EnVision as described in section Instrument setting.
10#Read plates on EnVision using FP Dual enhanced mirror, FP 480 excitation filter, FP-P-pol 535 and FP-S-pol 535 emissin filters

Comment: Actives were selected based on, % response = 45% or greater

External ID: HGDAF12_AG_LUMI_1536_1X%ACT PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as agonists of the nuclear receptor daf-12. In this assay, CV-1cells are co-transfected with a GAL4-responsive reporter plasmid (UAS-tk-luc) and expression vector encoding GAL4-hgDAF12. The ability of compounds to increase transcriptional activity is assessed by measuring luciferase expression from the reporter gene plasmid. Compounds were tested in singlicate at a final nominal concentration of 6.8 micromolar.

Protocol Summary:

HEK293 cells were routinely cultured in T-175 flasks containing 25 mL of DMEM media supplemented with 10% v/v fetal bovine serum and 1% v/v antibiotic-antimycotic mix at 37 C, 5% CO2 and 95% relative humidity (RH). The day prior to run the assay, the HEK293 cells were harvested using 5 mL of TrypLE reagents and seeded in fresh media at a density of 10 million cells per T175 flask. The following day, cells were transfected with 1 mL of serum-free OptiMEM containing 8 ug of the hgDAF12-expressing vector, 20 ug of the MH1000-tk-luc reporter plasmid and 80 uL of transfection reagent. Twenty four hours post transfection, cells were harvested using 5 mL of preheated TrypLE and resuspended at a concentration of 1 million cells per mL in phenol-red free DMEM supplemented as above. Sodium butyrate, a compound shown to increase the luminscent signal in this assay, was used as a positive control.

The assay was started by dispensing 5 uL of cell suspension into each well of white, solid-bottom 1536-well plates using a flying reagent dispenser (Aurora) and placed in the incubator for 3 hours. Cells were then treated with 34 nL/well of either test compounds, DMSO (Low Control, final concentration 0.68%) or 2 mM of Sodium Butyrate (High Control). Plates were incubated for 24 hours at 37 C, 5% CO2 and 95%RH and then removed from the incubator and equilibrated to room temperature for 10 minutes. Luciferase activity was detected by addition of 5 uL of One-Glo reagent to each well. After a 15 minute incubation time, light emission was measured with the ViewLux reader (PerkinElmer).

The percent activation of each test compound was calculated as follows:

%_Activation = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median__Low_Control ) )

Where:

High_Control is defined as wells treated with 2 mM Sodium Butyrate
Low_Control is defined as wells treated with DMSO only.
Test_Compound is defined as wells treated with test compound.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary activation. Negative % activation values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-12, and for inactive compounds 12-0.

List of Reagents:

MH1000-tk-luc luciferase reporter plasmid (Assay Provider)
hgDAF12 expressing plasmid (Assay Provider)

List of Consumables:

Sodium Butyrate (Sigma, part B5887)
HEK293 cells (ATCC, part CRL-1573)
DMEM (Invitrogen, part 11965)
FBS (Hyclone, part SH30088.03)
Antibiotic-Antimycotic 100X Liquid Solution (Gibco, part 15240)
TransIT 293 (Mirus Corporation, part MIR-2700)
OptiMEM (Invitrogen, part 31985)
TrypLE Trypsin Replacement Enzyme (Invitrogen, part 12604)
One-Glo (Promega, part E6130)
1536-well plates (Greiner part 789173)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate luciferase activity and hence well luminescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

In absence of a Protein Entry for the DAF-12 protein from H. glycines in NCBI's database, links to the target sequence and origin refer to the closest known orthologue from the prototypic parasitic helminth C.elegans.

External ID: TPD2_INH_EPIABS_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as inhibitors of the activity of tyrosyl DNA phosphodiesterase 2 (TDP2). TDP2 is a divalent cation-dependent enzyme that repairs TopII-associated DNA strand breaks. It is hypothesized that inhibitors of TDP2 may serve as useful adjuvants in combination with cancer drugs such as etoposide.

In this biochemical assay, recombinant human TDP2 protein is incubated at 37 degrees Celsius with the T5PNP substrate in the presence of Mg2+-containing assay buffer.T5PNP is a substrate for snake venom phosphodiesterase, as well as a substrate for TDP2. As a substrate for TDP2, T5PNP is used to mimic the TopII-DNA complex. TDP2 cleaves the phosphodiester bond in T5PNP, and the chromogenic p-nitrophenol group is released. As time increases the TDP2 enzyme will increasingly catalyze hydrolysis of the T5PNP substrate, resulting in increased release of p-nitrophenol and detection at 415nM wavelength. Compounds are tested in singlicate at a final nominal concentration of 12.8microM.

Protocol Summary:

Prior to the start of the assay, 2 ul of a solution containing T5PNP subtrate (final concentration 5mM) in assay buffer (50mM Tris-HCl pH7.5, 1mM DTT, 1mM MgCl2, 50mM KCl and 100ug/ml BSA) was dispended into a all wells of a 1536 well plate. Next, 39nL of test compound in DMSO or DMSO alone (1% final concentration) was added to the appropriate wells. The assay was started by dispensing 1 ul of a solution contanting TDP2 enzyme (120nM final concentration) in assay buffer to wells in columns 4-48 and 1ul of assay buffer alone to wells in columnes 1-3. Plates were centrifuged and incubated for 2hrs at 37 degrees Celsius at which time fluorescence intensity was measured (Ex. 405nm and Em. 405nm) using a EnVision microplate reader (Perkin Elmer).

Prior to further calculations, the following formula was used to calculate Epi Absorbance (EPIABS)

EPIABS = -log10( sample / background)

Where:

Sample is defined as the fluorescent intensity of wells containing test compounds or DMSO.
Background is defined as the fluorescent intensity of wells containing buffer and T5PNP only.

The % inhibition for each well was then calculated as follows:

%_Inhibition = ( EPIABS_Test_Compound - MedianEPIABS_Low_Control ) / ( MedianEPIABS_High_Control - MedianEPIABS_Low_Control ) * 100

Where:

Test_Compound is defined as wells containing test compound, TDP2 and T5PNP.
High_Control is defined as wells containing only Buffer and T5PNP.
Low_Control is defined as wells containing DMSO, TDP2 and T5PNP.

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

PubChem Activity Outcome and Score:

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-22, and for inactive compounds 22-0.

List of Reagents:

TDP2 (supplied by Assay Provider)
Tris Base(Sigma, 93349)
DTT (Sigma, 43815)
BSA (Sigma, A2153)
MgCl2 (Sigma,M2670)
KCl (Sigma, P9333)
T5PNP (Sigma, T4510)
1536 well plates (Corning 7254)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: HMS937_MLP

Protocol: 10 microL of proteasome-containing buffer (0.15 nM proteasome in 50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) were added to each well of 384-well plates (Corning 3820), except for column 1, which was used for positive controls.

100 nL of each experimental compound were transferred to the assay plates by pin transfer, and then incubated for 30 minutes at room temperature.

10 microL of 1 microM Ub-AMC in buffer (50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) were added to all wells of the assay plates, and then incubated for 25 minutes at room temperature. The fluorescence intensity of each well was then read using a Perkin Elmer EnVision plate reader at 355 nm/460 nm (excitation/emission).

For positive controls, 10 microL of 1 microM Ub-AMC in buffer (50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) were added to all wells in column 1 on each assay plate, and incubated for 25 minutes at room temperature before fluorescence reading.

For negative controls, 10 microL of proteasome-containing buffer (0.15 nM proteasome in 50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) were added to all wells in column 2 on each assay plate, and then 10 microL of 1 microM Ub-AMC in buffer (50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) was added. After incubation at room temperature for 25 minutes, fluorescence intensity was read.

Comment: Activity scores were calculated based on percent (%) inhibition, which was in turn calculated from fluorescence intensity (FI) readings as follows: % Inhibition = (1-((Vi-Vp)/(Vn-Vp)))*100, where Vi is the FI of the individual well, Vp the average FI of the positive controls on that plate, and Vn the average FI of the negative controls on that plate. A final % inhibition value was calculated as the average of replicate results for each individual compound and used to determine the Activity Score. If the % inhibition was less than zero, the Activity Score was set to Zero. Compounds showing >20% inhibition were considered active.

External ID: PLCG1_INH_QFRET_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this biochemical assay is to identify compounds that act as inhibitors of the activity of phospholipase C isozymes, PLC-G1. In this assay, PLC-G1 isozyme is incubated with test compounds and fluorogenic reporter WH-15. As designed, test compounds that act as PLC-G1 inhibitors will prevent the hydrolysis of WH-15 fluorogenic reporter, thus preventing the release of IP3, a quinomethide derivative, and 6-aminoquinoline, which is highly fluorescent, leading to decreasing well fluorescence. Compounds are tested in singlicate at a nominal test concentration of 12.2 micromolar.

Protocol Summary:

Prior to the start of the assay, 2 microliters of PLC-G1 at a final concentration of 5pg/ul (in 50 mM HEPES pH 7.2, 70 mM KCl, 3mM CaCL2, 3mM EGTA, 2mM DTT, 0.04mg/mL acid-free BSA, with Cholate 0.5%) are dispensed into 1536 microtiter plates, 1 microliter of assay buffer is dispensed into columns 4-48 and 1 microliter of 0.2M EGTA is added to columns 1-3. Compounds are added to plate (final concentration 12.2uM) and incubated for 10 minutes at 25 degrees Celsius. The assay start by the addition of 2 microliter of WH-15 fluorogenic reporter at a final concentration 10uM in Assay Buffer to all wells. Plates were centrifuged and after 90 min of incubation at 25 degrees Celsius fluorescence is measured at 355nm excitation and 535nm emmision.

The percent inhibition for each compound was calculated as follows:

100 *( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )

Where:

Test_Compound is defined as wells containing PLCG1 in the presence of test compound and WH15 fluoreogenic reporter.
High_Control is defined as wells containing PLCG1, WH15 fluoreogenic reporter and EGTA.
Low_Control is defined as the median of the wells containing test compounds.

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Four values were calculated: (1) the average percent inhibition of all high controls tested plus three times the standard deviation of the high controls, (2) the average percent inhibition of all low controls tested minus three times the standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values, (3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity than the cutoff parameter was declared active.

PubChem Activity Outcome and Score:

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The activity score range for active compounds is 100-11, for inactive 11-0.

List of Reagents:

PLCG1 isozyme (Supplied by Assay Provider)
WH-15 fluorogenic reporter (Supplied by KXTBio)
HEPES (Fisher, BP310)
Sodium cholate hydrate (Sigma, C6445)
CaCl2 (Sigma, 06991)
EGTA (Fisher, O2783)
DTT (Fisher, BP172)
KCl (Sigma, P9541)
1536-well plates (Corning, part 7261)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: JHICC_RGS_Inh_HTS

Protocol: Assay overview:

To screen for compounds that inhibit the RGS4 protein, a HEK293 cell line which stably expresses M3R and inducibly expresses RGS4 is employed. RGS4 function is monitored by calcium flux with a commercially available Fluo4-AM dye. Compounds that show increase in the Fluo4 fluorescence in induced RGS4 expressed cells are considered agonist hits. M3 receptor and other endogenous receptor inhibitors will be excluded through later counter-screening against non-induced parental cells.

Protocol for RGS4 Primary Screen:
1. Cell culture: Cells (HEK293-FlpIn-TREx/M3R/RGS4) are routinely cultured in DMEM (high glucose, w/ glutamine), 10% FBS, 1%Pen/Strep, 15 ug/ml Blasticidin, 400 ug/ml G418, 200 ug/ml Hygromycin.
2. Cell plating: Add 50 ul/well of 200,000 cells/ml re-suspended in DMEM/high glucose medium with 10% FBS, 1%Pen/Strep. Include 10 ng/ml Doxycyclin (DOX) to induce RGS4 expression.
3. Incubate overnight at 37 degrees C and 5% CO2.
4. Remove medium and add 20 ul /well of 2 uM Fluo4-AM solution to cells.
5. Incubate 30 minutes at 37 degrees C in incubator.
6. Prepare 6x compound plates and control plates on Cybi-Well system: test compounds are prepared using assay buffer (HBSS-HEPES pH 7.4).
7. Remove Fluo4-AM dye solution and add 20 ul /well of assay buffer to cells.
8. Incubate 30 minutes at room temperature (RT).
9. Add 6x compounds in cell plates and incubate 20 minutes at RT.
9. Load cell plates on Hamamatsu FDSS 6000 kinetic imaging plate reader
10. Measure fluorescence for 10 seconds at 1Hz to establish baseline
11. Add 4 ul of 7x EC20 (carbachol) into the cell plates and record fluorescence for 100 seconds.
12. Calculate ratio readout as F(max-min)/F0 and integrated ratio readout.
13. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z prime factors [26]
14. Calculate B scores [27] for test compounds using integrated ratios calculated in Step 12
15. Outcome assignment: If the B score of the test compound is more than 3 times the standard deviation (SD) of the B scores of integrated ratios of all library compounds above the mean (B score ratio>3*SD+mean), AND the ratio of initial fluorescence intensity is within 5 times the standard deviation plus or minus the mean of the ratios of the library compounds, the compound is designated in the Outcome as active (value=2) as an inhibitor of RGS4. Otherwise, it is designated as inactive (value=1).
16. Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of Int(((Log(ABS(B score ratio))-0.6)/1.25)*100), they are normalized to the smallest and largest LOG(B score ratio), B score ratio, as in the result definition. The inactive test compounds are assigned a score of 0.

List of reagents

1. HEK293-FlpIn-TREx/M3R/RGS4 cell lines (provided by assay provider)
2. PBS: pH7.4 (Invitrogen Catalog number 10010049)
3. Medium: DMEM (Sigma, Catalog number D5796)
4. Fetal Bovine Serum (Gemini, Catalog number 100-106)
5. Hygromycin (Mediatech, Catalog number 30-240-CR)
6. 100x Penicillin-Streptomycin (Mediatech, Catalog number 30-001-CI)
7. Cell/stripper (Mediatech, Catalog number 25-056-Cl)
8. G418: (Invitrogen, Catalog number 11811-031)
9. Blasticidin (Sigma, Catalog number R21001)
10. Doxycycline hyclate (Sigma, Catalog number D9891)
11. HEPES (Sigma, Catalog number H4034)
12. Fluo-4 (Invitrogen, Catalog number F14202)
13. Pluronic F-127*20% in DMSO (Invitrogen, Catalog number P-3000MP)
14. Atropine (Sigma, Catalog number A0132)
15. Carbachol (Sigma, Catalog number C4382)
16. Triple-layer flask (VWR, Catalog number 62407-082)
17. BD Biocoat 384-well plates (BD, Catalog number (35)4663 and Lot number 7346273)

Comment: Possible artifacts of this assay can include, but are not limited to: non-intended chemicals or dust in or on wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, and compounds that quench or emit light or fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.

External ID: HMS1364-MLP_WT

Protocol: The day before screening, RN4220 was grown overnight (16-18 h) in sterile-filtered tryptic soy broth (TSB) at 30 degrees C with shaking.

On the day of screening, columns 1-24 of assay plates (Corning 3710, clear) were prefilled with 30 uL of TSB using the Combi. Next, 100 nL of each compound was pin-transferred to each plate. For every compound library plate, there were two daughter plates (A & B). Overnight cultures were diluted 625x into TSB. 50 uL of the diluted cultures were added to columns 1-24 of the corresponding assay plates. Wells in column 2 contained only medium (with culture) and served as the negative controls. Positive control (0.8 uL of erythromycin, 10 ug/mL) was added to column 1 using the Hewlett Packard D300. Final assay well volume was 80 uL. Plates were spun down and stacked 5 high, covered with lids (Corning 3009), and incubated at 30 degrees C overnight (~16 h).

The following day, assay plates were removed from the incubator and cell density quantitated using a PerkinElmer EnVision (600 nm filter).

Comment: Z-scores were calculated for every well based on plate average and standard deviation of experimental well absorbance (OD600). Wells were considered active for reduced WT survival if Z-score for both replicates <= -3 and absorbance for both replicates <= 0.18. Percent inhibition was calculated by subtracting well absorbance from negative control plate average absorbance, dividing by the difference between plate negative and positive control plate average absorbance, and multiplying by 100. The replicate values were then averaged to determine activity scores. Resulting values > 100 were set to 100 and < 0 were set to 0, where 100 = 100% activity.

External ID: FLUC100

Protocol: NCGC Assay Protocol Summary:

Reagents: 50mM Tris acetate, pH 7.5; 10mM Mg acetate; 10uM D-luciferin (Sigma #L9504); 10uM ATP; 0.01% Tween-20; 0.05% BSA; 10nM P. pyralis luciferase (Sigma #L9506)
Control compounds used were two known firefly luciferase inhibitors (compounds (2) and (5) in Auld et al., 2010), and DMSO.

Assay Summary:
Three microliters containing firefly luciferase substrates in buffer (final concentrations: 50mM Tris acetate, pH 7.5, 10mM Mg acetate, 0.01% Tween-20, 0.05% BSA, 10uM D-luciferin, and 10uM ATP) are dispensed into each well of a Greiner white, solid-bottom 1536-well format plate using a flying reagent dispenser (FRD). These assay plates were then treated with 23nL of compound or DMSO using a Kalypsys pin tool, which allows for delivery of a 6-point interplate titration of each compound to the assay plate (quantitative HTS), with a final compound concentrations ranging from approximately 60muM to 7pM. One microliter of firefly luciferase in 500mM Tris-acetate buffer was then delivered by FRD to each well for a final enzyme concentration of 10nM. Luciferase activity was then measured using a ViewLux CCD imager (PerkinElmer), with an average exposure time of 2-30 seconds (2X binning, medium/high gain).

Keywords: NIH Roadmap, MLPCN, MLSMR, qHTS, miR-21, firefly luciferase, FLuc, miRNA.

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: HMS1364-MLP_ugtP

Protocol: The day before screening, RN4220 ugtP was grown overnight (16-18 h) in sterile-filtered tryptic soy broth (TSB) at 30 degrees C with shaking.

On the day of screening, columns 1-24 of assay plates (Corning 3710, clear) were prefilled with 30 uL of TSB using the Combi. Next, 100 nL of each compound was pin-transferred to each plate. For every compound library plate, there were two daughter plates (A & B). Overnight cultures were diluted 625x into TSB. 50 uL of the diluted cultures were added to columns 1-24 of the corresponding assay plates. Wells in column 2 contained only medium (with culture) and served as the negative controls. Positive control (0.8 uL of erythromycin, 10 ug/mL) was added to column 1 using the D300. Final assay well volume was 80 uL. Plates were spun down and stacked 5 high, covered with lids (Corning 3009), and incubated at 30 degrees C overnight (~16 h).

The following day, assay plates were removed from the incubator and cell density quantitated using a PerkinElmer EnVision (600 nm filter). Library plates were screened in duplicate, with both assay plates in a given set prepared on the same day.

Comment: Z-scores were calculated for every well based on plate average and standard deviation of experimental well absorbance (OD600). Wells were considered active for reduced ugtP survival if Z-score for both replicates < -3 and absorbance for both replicates <= 0.15. Percent inhibition was calculated by subtracting well absorbance from negative control plate average absorbance, dividing by the difference between plate negative and positive control plate average absorbance, and multiplying by 100. The replicate values were then averaged to determine activity scores. Resulting values > 100 were set to 100 and < 0 were set to 0, where 100 = 100% activity.

External ID: TTR_ACT_LUMI_1536_1X%ACT PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify new small molecules which specifically enhance the synthesis of neuronal Transthyretin (TTR) transcription without increasing hepatic or peripheral TTR levels. In this assay, SHSY5Y human neuroblastoma cells stably expressing the Gaussia luciferase (eGLuc2) gene under the control of 2kb of the human TTR promoter (SHSY5Y TTR eGLuc2) are incubated with test compounds for a defined period, followed by the addition of luciferase substrate followed by a luminescence measurement. As designed, test compounds that activate neuronal TTR levels, will increase well luminescence. Compounds are tested in singlicate at a final nominal concentration of 16.7 uM.

Protocol Summary:

The stably transfected SHSY5Y-TTR neuroblastoma cells were routinely cultured in T-225 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of DMEM supplemented with 10% v/v Fetal Bovine Serum, 500ug/mL Geneticin and 1X Penicillin-Streptomycin-Glutamine.

Prior to assay, cells were suspended to a concentration of 500,000 cells/mL in DMEM containing 10% v/v Fetal Bovine Serum and 1X Penicillin-Streptomycin-Glutamine. The day before the assay, 3uL of Recombinant Lucia Luciferase Protein (20ng/mL final concentration) was dispensed into column 3 only and 1500 cells in 3 uL of cell assay media were seeded into the remaining wells. Next, 51 nL of test compound in DMSO, F11 or FD (167 uM final concentration) in DMSO, or DMSO alone (1.67% final concentration) were dispensed to the appropriate wells. The plates were then incubated for 24 hours at 37 C, 5% CO2, and 95 % RH.

Following the one day incubation, plates were equilibrated to room temperature for 10 minutes. The assay was started by adding 3 uL of QUANTI-Luc detection reagent (prepared according to the manufacturer's protocol) to all wells. Plates were centrifuged and well luminescence was read on a ViewLux microplate reader (Perkin Elmer, Turku, Finland).

The percent activation for each compound was calculated as follows:

%_Activation = 100 * ( ( RLU_Test_Compound - Median_RLU_Low_Control ) / ( Median_RLU_High_Control - Median_RLU_Low_Control ) ) )

Where:

Test_Compound is defined as wells containing cells, test compounds and DMSO.
High_Control is defined as wells containing LUCIA and DMSO.
Low_Control is defined as the median of the wells containing test compounds.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated: (1) the average percent activation of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % activation than the cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The activity score range for active compounds is 100-7, for inactive 7-0.

List of Reagents:

TTR-SHSY5Y cells (supplied by Assay Provider)
DMEM (Cellgro, part 10-013-CV)
Fetal Bovine Serum (Cellgro, part 35-010-CV)
Penicillin-Streptomycin-Glutamine (100X) (Life Technologies, part 10378-016)
Recombinant Lucia Luciferase Protein (Invivogen, part rec-lucia)
QUANTI-Luc Detection Reagent (Invivogen, part rep-qlc)
F11 (Activator Reference Control, Fisher-Maybridge, part SPB05942SC)
FD (Inhibitor Reference Control, Sigma, part F8257)
Trypsin-EDTA (Life Technologies, part 25200-114 )
Geneticin (Life Technologies, part 10131-027)
T-225 Flasks (BD, part 353138)
1536-well plates (Greiner, part 789173)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: 7056-01_Inhibitor_SinglePoint_HTS_Activity

Protocol: Bac rRNA Automation Protocol
Day 1:
1. Grow each E. coli strain (one wild type (GFP) and six humanized mutants (RFP) including the control G2058) individually. Pick each strain into a separate 50 mL conical tube with 10 mL of LB medium supplemented with 100 ug/mL amplicillin and 50 ug/mL spectinomycin.
2. Incubate strains overnight at 37 degrees with shaking at 190 RPM.
Day 2:
1. In the morning, check OD600 of the cultures.
2. Dilute the cultures to OD 600 = 0.05 in LB medium supplanted with 100 ug/mL ampicillin.
3. Return strains to 37 degrees with shaking at 190 RPM for about 3. 5 hours. Grow strains to reach OD 600 about 0.5 (exponential growth phase).
4. Mix strains (final OD600= 0.02) in the proportion 50% wild-type, 8.33% each of the six mutants in LB medium supplemented with 100 ug/mL ampicillin and 1 mM IPTG (which is used to induce expression of fluorescent proteins).
5. Dispense 30 uL/well of bacteria mixture to 384 well Assay Ready Plates (ARPs) containing positive controls (Erythromycin 15 mg/mL, 50 nL in column 1 and Choramphenicol 15 mg/mL, 50 nL in column 23).
6. Place breathable film on plate using Plate Loc.
7. Incubate plates in Steristore at 37 degrees, 0% CO2 for 24 hours, 95% humidity.
Day 3:
1. Remove assay plate from Steristore.
2. Remove breathable film with X-Peel.
3. Read GFP signal using EnVision plate reader (mirror: FITC #403, Ex: FITC 485 #102, Em: FITC 535 #206).
4. Read RFP signal using EnVision plate reader (mirror: BODIPY TMR #405, Ex: photometric 550 #312, Em: Cy3 595 #229).
5. Read OD600 signal using EnVision plate reader (Ex: Photometric 600 #319).
6. Store plate in Steristore incubator.
Equipment Required
Combi
Plate Loc
Steristore (37 C, 95% humiodity, 0% CO2,)
X-Peel
2 EnVisions

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control
wells (PC) were included on every plate. One positive control,
Chloramphenicol, was used to determine the baseline fluorescence and OD
as it was expected to non-selectively kill all 6 strains, GFP wild-type
and RFP mutant. The other positive control, Erythromycin, was expected
to selectively spare one RFP control strain (G2058) and was therefore
used as a guideline for calculating the performance of a selective GFP
inhibitor.

NORMALIZATION:
Each of three layers was independently normalized using the 'Neutral
Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to
a normalized activity value of 0.
The a raw signal of 0 was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: The plate pattern correction algorithm 'Assay
Pattern (multiplicative)' in Genedata (v7.0.3) was applied to the
normalized plate data for each layer.

AGGREGATE COMPOUND LAYER SCORING: In addition to the reported normalized
percent activities above, each replicate compound measurement was
converted to a z-score relative to the corresponding DMSO-control
distribution, as described for ChemBank (Seiler et al., 2008). ChemBank
composite Z scores were computed to combine replicates for each channel
(RFP, GFP, and OD). We sought compounds that affected the GFP-expressing
wild-type strain while not affecting at least one of the mutant RFP
strains and not affecting overall viability as measured by OD; i.e.,
they score with negative composite Z in the GFP channel with little or
no effect in the RFP or OD channels. To combine the results of the three
channels quantitatively, we defined a trigonometric scaling function to
express our preferences that (1) GFP composite Z scores be negative, and
(2) RFP and OD composite Z score amplitudes exceed GFP composite Z score
amplitudes; such compounds should fall left of the DMSO-control
distribution, but above the main diagonal in a plot of GFP composite Z
versus either OD or RFP composite Z. Using the two ratios of ChemBank
composite Z scores (GFP/RFP and GFP/OD), we defined q1 = arctan(GFP/RFP)
and q2 = arctan(GFP/OD) and expressed our scale factor as F =
cos(-q1)cos(2q1)cos(-q2)cos(2q2), with the four factors respectively
expressing our preferences. As a three-assay composite score, we take
-(ZGFP) x F, which clearly discriminates the compounds of interest when
plotted against RFP or OD.

PUBCHEM_ACTIVITY_SCORE: The above three-assay composite score was
normalized on a scale of 1-100, with 1 being those with the least
desirable observed score representing compounds increasing in GFP signal
and decreasing in RFP or OD, and 100 being the most ideal observed score
representing a loss in GFP with no change in RFP or OD.

PUBCHEM_ACTIVITY_OUTCOME: The above three-assay composite scores were
fit to a standard distribution and a corresponding Holm-Bonferroni
corrected p value significance was calculated. Compounds with a FDR
corrected p value less than 0.05, normalized OD percent score > -30,
negative GFP composite Z score, and the expression Abs(Cos(-q)Cos(2q)) >
(sqrt2)/2 for both q1 and q2 were assigned a score of 2 (active).
Compounds failing any of these criteria were assigned a score of 1
(inactive.)

External ID: PLCB3_INH_QFRET_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this biochemical assay is to identify compounds that act as inhibitors of the activity of phospholipase C isozymes, PLC-B3. In this assay, PLC-B3 isozyme is incubated with test compounds and fluorogenic reporter WH-15. As designed, test compounds that act as PLC-B3 inhibitors will prevent the hydrolysis of WH-15 fluorogenic reporter, thus preventing the release of IP3, a quinomethide derivative, and 6-aminoquinoline, which is highly fluorescent, leading to decreasing well fluorescence. Compounds are tested in singlicate at a nominal test concentration of 12.2 micromolar.

Protocol Summary:

Prior to the start of the assay, 2 microliters of PLC-B3 at a final concentration of 0.4ng/ul (in 50 mM HEPES pH 7.2, 70 mM KCl, 3mM CaCL2, 3mM EGTA, 2mM DTT, 0.04mg/mL acid-free BSA, with Cholate 0.5%) are dispensed into 1536 microtiter plates, 1 microliter of assay buffer is dispensed into columns 4-48 and 1 microliter of 0.2M EGTA is added to columns 1-3. Compounds are added to plate (final concentration 12.2uM) and incubated for 10 minutes at 25 degrees Celsius. The assay start by the addition of 2 microliter of WH-15 fluorogenic reporter at a final concentration 10uM in Assay Buffer to all wells. Plates were centrifuged and after 90 min of incubation at 25 degrees Celsius fluorescence is measured at 355nm excitation and 535nm emmision.

The percent inhibition for each compound was calculated as follows:

100 *( ( Test_Compound - Median_Low_Control) / ( Median_High_Control - Median_Low_Control) )

Where:

Test_Compound is defined as wells containing PLCB3 in the presence of test compound and WH15 fluoreogenic reporter.
High_Control is defined as wells containing PLCB3, WH15 fluoreogenic reporter and EGTA.
Low_Control is defined as the median of the wells containing test compounds.

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Four values were calculated: (1) the average percent inhibition of all high controls tested plus three times the standard deviation of the high controls, (2) the average percent inhibition of all low controls tested minus three times the standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values, (3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity than the cutoff parameter was declared active.

PubChem Activity Outcome and Score:

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The activity score range for active compounds is 100-13, for inactive 13-0.

List of Reagents:

PLCB3 isozyme (Supplied by Assay Provider)
WH-15 fluorogenic reporter (Supplied by KXTBio)
HEPES (Fisher, BP310)
Sodium cholate hydrate (Sigma, C6445)
CaCl2 (Sigma, 06991)
EGTA (Fisher, O2783)
DTT (Fisher, BP172)
KCl (Sigma, P9541)
1536-well plates (Corning, part 7261)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: ADAM10_INH_QFRET_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that inhibit ADAM10. This assay employs a fluorophore and quencher pair. F =EDANS fluorophore, Q = DABCYL quencher. When intact, EDANS emission at 460nm is quenched by DABCYL via fluorescence resonance energy transfer. Upon cleavage of the scissile bond (A~V) by ADAM protease, the distance between fluorophore and quencher increases resulting in fluorescence increase at 460nm. Compounds are tested in singlicate at a nominal final nominal concentration of 6.95 micromolar.

Protocol Summary:

Prior to the start of the assay, 2.5 microliters 2X ADAM10 enzyme (20 nM in Assay Buffer: 50 mM HEPES, 0.01% Brij, pH 7.5) are dispensed into 1536 microtiter plates. Compounds are added to plate (final concentration TBD) and incubated for 30 minutes at 25 degrees Celsius. The assay is started by dispensing 2.5 microliter of2X DM2 substrate (20 uM in Assay Buffer) to all wells. Plates are centrifuged and after 3 hours of incubation at 25 degrees Celsius, fluorescence is measured (excitation = 359nm, emission = 460nm).

The % inhibition for each well was then calculated as follows:

%_Inhibition = ( RFU_Test_Compound - MedianRFU_Low_Control ) / ( MedianRFU_High_Control - MedianRFU_Low_Control ) * 100

Where:

Test_Compound is defined as wells containing test compound.
High_Control is defined as wells treated with 1 micromolar Marimastat
Low_Control is defined as wells containing DMSO.

Interval Cutoff:

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Four values were calculated: (1) the average percent inhibition of all high controls tested plus three times the standard deviation of the high controls, (2) the average percent inhibition of all low controls tested minus three times the standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values, (3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity than the cutoff parameter was declared active.

PubChem Activity Outcome and Score:

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The activity score range for active compounds is 100-19, for inactive 19-0.

List of Reagents:

ADAM10 enzyme (R&D Systems, part # 936-AD)
EDANS-DABCYL DM2 peptide substrate (Supplied by Assay Provider)
0.5M HEPES solution, pH7.5 (Teknova, part #101319-900)
Brij-35 (Sigma-Aldrich, part # P1254)
1536 well plate (Corning, part # 7261)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: SBCCG-A413-tim10-1-Primary-Antagonist-Assay

Protocol: Assay Materials:
Yeast Strain: ySHDSTim10ts
Growth Media: YPD broth (TEKNOVA)
SD Assay Media: 1.2 g/L Yeast Nitrogen Base w/o amino acids, 5 g/L Ammonium Sulfate, 20 g/L Dextrose, 13.8 g/L Succinate, supplemented with 1 X Amino Acid Mix (2 g/L) (Sunrise Science Products, San Diego, CA)
SD Minimal Media: 1.2 g/L Yeast Nitrogen Base w/o amino acids, 5 g/L Ammonium Sulfate, 13.8 g/L Succinate, (Sunrise Science Products, San Diego, CA)
Assay Plate: Corning 1536 Well White Plate (Catalogue #: 3725)
Detection Reagent: Bactiter-GLO (Promega)

I. Compound Addition:

1. Using LabCyte Echo, transfer 40 nL from a 2 mM Echo qualified plate containing test compounds into assay plate columns 3 - 48 (final concentration of test compounds is 20 uM, 1 % DMSO). Transfer 40 nL of DMSO to positive and negative control wells in columns 1 - 4.
2. Centrifuge plates at 1000 rpm for 1 min.

II. Set up of tim10-1 assay:

The day before the screen, frozen culture was thawed at room temperature and resuspended in growth media at a cell density of 5x10^5/ml in approx. 100 ml. The culture was grown overnight at 25 oC with shaking (225 rpm).

3. In the morning of the Set-Up day prepare sufficient amount of SD Assay Media for negative control and compound wells and SD Minimal Media for positive control wells in order to obtain enough yeast cell culture for plating the desired number of 1536 well plates with 4 ul yeast cell suspension / well.
4. Count the yeast cells from the overnight Growth Media culture.
5. Dilute yeast to a final concentration of 2000 yeast/well in SD Media.
6. Pellet at 2400 rpm for 5 min at RT. Aspirate off supernatant.
7. Add 25 ml of sterile Water. Re-suspend cells by gently shaking. Pellet again at the same conditions, and wash the yeast cells in 25 ml sterile water a second time.
8. Re-suspend in SD Assay Media for negative control and compound wells.
9. For positive control wells, use SD Minimal Media with no yeast.
10. Add 4 ul yeast cells per well using combi and cover each plate with plastic lid.
11. Spin the plates at 2000 rpm for 1 min, incubate at 25 oC, inverted in a stack of 4, wrapped in saran wrap for 22-24 hours.

IV. Reading plates:
12. After 22-24 hours of incubation, add 3 ul of substrate solution (should be at RT) to all the wells of each plate using combi.
13. Plates are spun again at 2000 rpm, and left at RT for 15 min.
14. Read plates using a Perkin Elmer ViewLux using a luminescence protocol.

Comment: Compounds with %Activity >= 40% are defined as actives in this assay.

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary and single-concentration confirmation screening data.
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30.
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.

2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay

External ID: MH083223 Targeting HIV-1 Nef with Small Molecules

Protocol: HIV Hck:Nef HTS Protocol
1. Recombinant Hck-YEEI protein, was purified to homogeneity from Sf-9 insect cells
2. Recombinant HIV-1 Nef (SF2 strain), was purified to homogeneity from E. coli
3. Z-Lyte assay reagents were purchased from (Invitrogen).

Several Hck and Nef protein preparations provided by the assay provider were utilized in the HTS campaign, and several Hck:Nef protein ratios were utilized 10 ng + 50 ng, 12.5 ng + 75 ng, 15 ng + 75 ng, and 20 ng + 100 ng in the 384-well HTS assay.

HTS Protocol:
1. Thaw compound plates, 2uL of 1 mM in 100% DMSO.
2. Spin compound plates down, 5 min 50 x g.
3. Dilute 2 uL 1mM compounds with 23ul of water on Flex Drop (80 uM, 8% DMSO).
4. Mix and Transfer 2.5ul of compound to assay plate on EP3.
5. Transfer 5ul of Max (2.5ul DMSO and 2.5ul Hck:Nef) and Min (2.5ul DMSO and 2.5ul Hck/1x kinase) controls (from pre-made control plates) to assay plate on EP3.
6. Add 2.5ul of Hck:Nef to assay plate(340 wells), spin down and incubate for 30 min at ambient temperature (40 uM, 4% DMSO).
7. Add 5 uL of ATP/Tyr complex to whole plate on MicroFlow, spin down plates and put on shaker to incubate at ambient temperature for 50 min (20 uM, 2% DMSO).
8. Add Development buffer to whole plate on MicroFlow, spin down plates and put on shaker to incubate at ambient temperature for 60 min.
9. Add Stop reagent to whole plate on MicroFlow, spin down.
10. Read Donor to Acceptor FRET Emission Ratio on M5 plate reader within 2 hrs. Excitation of the donor fluorophore at 400 nm, Donor to Acceptor FRET Ratio = ratio of Coumarin emission 445 nm to Fluorescein emission 520 nm.

Comment: Hck-Nef Assay HTS Activity scoring rules:
The 384-well format Hck-Nef inhibitor HTS run at the PMLSC utilized % inhibition calculated from maximum (n=32) and minimum (n=24) plate controls, with a hit criteria of >/= 50% inhibition to identify active compounds.
Max control: 2.5ul DMSO and 2.5ul Hck:Nef
Min control: 2.5ul DMSO and 2.5ul Hck/1x kinase (No Nef)
Hck-Nef Inhibitor scoring rules:
PUBCHEM_ACTIVITY_OUTCOME
1 - Substance is considered inactive when the % inhibition is < 50 %
2 - Substance is considered active when % inhibition is >/= 50 %
3 - Substance activity outcome is inconclusive
PUBCHEM_ACTIVITY_SCORE
0-40 scoring range is reserved for primary HTS data
a) if the % inhibition is >/= 50 %, the score is 40.
b) if the % inhibition is < 50 %, the score is 0.

External ID: SBCCG-A539-APOBEC3A-Inhibitor-Primary-Assay

Protocol: Assay Materials:

Protein dilution buffer: Tris (pH 7.4) at 15 mM, NaCl at 150 mM, Triton X-100 at 0.5%, glycerol at 10%
Oligo dilution buffer: TE at 20 mM
Oligo: 5' d FAM-AAATATCCCAAAGAGAGA-TAMRA 3': BioSearch Technologies
UDG: New England Biolabs
A3A-MycHis: University of Minnesota
1N NaOH
2M Tris (pH 7.9)
Assay plate: Corning 1536 Well Black Solid Bottom Plate (Catalogue # 3724)

I. Compound Addition:
1- Using LabCyte Echo, transfer 40 nL from 2 mM compound source plate into assay plate Columns 5-48 (final concentration of test compounds is 20 uM, 1.0% DMSO), and 40 nL of DMSO to control wells in Columns 1-4.

II. Preparing reagents:
2- Prepare protein dilution buffer
3- Prepare oligo dilution buffer
4- Prepare intermediate A3A at 0.4 ng/ul. The final concentration of A3A in the plate is 0.2 ng/ul.
5- Prepare Oligo/UDG intermediate with 1 uM for Oligo and 0.002U/ul for UDG. The final concentration in the plate is 0.5 uM for Oligo and 0.001U/ul for UDG.
III. Reagent Addition
6- Add 2 ul of protein dilution buffer in columns 1 & 2
7- Add 2 ul of A3A in columns 3 to 48
8- Add 2 ul of Oligo/UDG to all columns
9- Cover the plate with Kalypsis metal lid; incubate the plate at room temperature for 45 minutes.
10- Add 1 ul of 1N NaOH to all columns
11- Cover the plate with Kalypsis metal lid; incubate the plate at room temperature for 30 minutes.
12- Add 1 ul of 2M Tris (pH 7.9) to all columns
13- Cover the plate with Kalypsis metal lid; incubate the plate at room temperature overnight.
IV. Reading plates:
14- Read the plate on PerkinElmer-EnVision plate reader (485/535)

Comment: Compounds that demonstrated an activity of >= 40% at 20 uM concentration and <= 30% at 20 uM in "uHTS identification of APOBEC3G DNA Deaminase Inhibitors via a fluorescence-based single-stranded DNA deaminase assay" (AID TBD)are defined as actives in this assay.

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.

2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay

External ID: COUPTFII_INH_LUMI_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this cell-based assay is to identify compounds that can inhibit COUP-TFII transcriptional activity. The expression vectors for COUP-TFII (pcDNA6.2-COUP-TFII) and the luciferase reporter NGFIA-Luc (pXP2-168) are transiently cotransfected into HEK-293T cells.

COUP-TFII has been shown to efficiently activate NGFI-A-Luc expression (17) and the readout can be measured by a luminometer. Small molecules that inhibit COUP-TFII transcriptional activity will decrease the promoter activity that can be detected by luciferase assay. As designed, compounds that inhibit COUP-TFII activity will decrease luciferase activity, resulting in decreased well luminescence.

Protocol Summary:

HEK293 cells were routinely cultured in T-175 flasks containing 25 mL of DMEM media supplemented with 10% v/v fetal bovine serum and 1% v/v antibiotic-antimycotic mix at 37 C, 5% CO2 and 95% relative humidity (RH). The day prior to run the assay, the HEK293 cells were harvested using 5 mL of TrypLE reagents and seeded in fresh media at a density of 10 million cells per T175 flask. The following day, cells were transfected with 1 mL of serum-free OptiMEM containing 12 ug of the plasmid encoding for COUP-TF2, 8 ug of the pX2-168 luciferase reporter plasmid, and 60 uL of X-tremeGene 9 transfection reagent. Twenty four hours post transfection, cells were harvested using 5 mL of preheated TrypLE and resuspended at a concentration of 800,000 cells per mL in phenol-red free DMEM supplemented as above. In the absence of a pharmacological positive control, COUP-TF2 inhibition was mimicked by transfecting cells in absence of the COUP-TF2 expressing vector (i.e. reporter plasmid only).

The assay was started by dispensing 5 uL of cell suspension into each well of white, sterile, solid-bottom 1536-well plates. The first three columns received control cells (no COUP-TF2 expressed) whereas the rest of the plate was dispensed with COUP-TF2-transfected cells. After addition of cells the plates were spun down. The plates were incubated for 4 hours and then treated with 43 nL/well of test compounds or DMSO (final concentration 0.65%) on COUP-TF2 cells and Control cells. Plates were incubated for eighteen hours at 37 C, 5% CO2 and 95% RH. Plates were then removed from the incubator and equilibrated to room temperature for 10 minutes. Luciferase activity was detected by addition of 5 uL of One-Glo reagent to each well. After a 10 minute incubation time, light emission was measured with the ViewLux reader (PerkinElmer).

The percent inhibition for each compound was calculated as follows:

%_Inhibition = ( ( Test_Compound - Median_Sample_Field ) / ( Median_High_Control - Median_Sample_Field ) ) * 100

Where:

High_Control is defined as wells containing cells transfected with reporter plamid only (pX2-168)
Test_Compound is defined as well containing cells cotransfected with pcDNA6.2-COUP-TFII and pX2-168 in the presence of test compounds
Sample_Field is defined as wells containing cells cotransfected with pcDNA6.2-COUP-TFII and pX2-168 in the presence of test compounds

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated for each assay plate: (1) the average percent inhibition of test compound wells that are within the High and Low Controls (i.e. higher than the average plues three standard deveiations of the Low Control wells and lower than the average minus three stardard deviation of the High Control wells) and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % inhibition than that particular plate's cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-34, and for inactive compounds 34-0.

List of Reagents:

pX2-168 luciferase reporter plasmid (Assay Provider)
pcDNA6.2-COUP-TFII plasmid (Assay Provider)
HEK293 cells (ATCC, part CRL-1573)
DMEM (Invitrogen, part 21063)
FBS (Hyclone, part SH30088.03)
Antibiotic-Antimycotic 100X Liquid Solution (Gibco, part 15240)
X-tremeGENE 9 DNA Transfection Reagent (Roche, part 06365809001)
OptiMEM (Invitrogen, part 31985)
TrypLE Trypsin Replacement Enzyme (Invitrogen, part 12604)
OneGlo (Promega, part E6130)
1536-well plates (Corning, part 7298)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: BCCG-A235-SUMO-HTRF-Assay

Protocol: Assay materials:
1) E1, E2, GST-SUMO and His6-RanGap-1 proteins were obtained from the assay provider's laboratory.
2) Assay Buffer: 50mM Tris-HCl pH 7.4, 0.3mM DTT, 10 mM MgCl2, 0.005% Tween-20
3) Anti-GST XL665 and Anti-His Europium-labeled antibodies were purchased from Cis-Bio (Cat # 61GSTXLB and 61HISXLB)
4) Corning 1536-well, white plates (Cat #3725)

uHTS Procedure
1) Dispense 2ul of assay buffer into columns 1 and 2, and 2ul of Mixture 1, containing 37.5 nM E1 and 100 nM His-RanGap-1 in the assay buffer, into columns 3-48 of the 1536-well assay plate.
2) Using a HighRes biosolutions pintool dispense 70nl of 2mM compounds in DMSO to columns 5-48
3) Dispense 70nl of DMSO to columns 1-4.
4) Using Thermo Scientific MultiDrop Combi, dispense 2 uL of Mixture 2, containing 20 mM ATP, 12.5 nM E2 and 30 nM GST-SUMO in assay buffer, to all wells.
5) Incubate for 30 min. at room temperature.
6) Add 1ul of 500 mM KF
7) Read plate on a BMG Labtech PheraStar in a Homogeneous Time-Resolved
Fluorescence mode (Ex: 337 nm; Em: 620/665 nm). The ratio of fluorescence 665 over 620 was utilized as a readout of the assay.
8) Data analysis was performed using CBIS software (ChemInnovations, Inc).
9) The value of fluorescence at 620 nm for each sample was normalized to the value of the average value of fluorescence at 620 nm in the plate negative control wells to calculate F_ratio parameter.

Procedure for dose-response confirmation assay

1) Using Thermo Scientific MultiDrop Combi, dispense 2ul of assay buffer into columns 1 and 2, and 2ul of Mixture 1, containing 37.5 nM E1 and 100 nM His-RanGap-1 in the assay buffer, into columns 3-48 of the 1536-well assay plate.
3) Using Labcyte Echo550, dispense 70 nL of serially diluted compounds to columns 5-48 and DMSO to columns 1-4.
4) Using Thermo Scientific MultiDrop Combi, dispense 2 uL of Mixture 2, containing 20 mM ATP, 12.5 nM E2 and 30 nM GST-SUMO in assay buffer, to all wells.
5) Incubate lidded plate for 30 min. at room temp.
6) Add 1ul of 500 mM KF
7) Read plate on a BMG Labtech PheraStar in a Homogeneous Time-Resolved
Fluorescence mode (Ex: 337 nm; Em: 620/665 nm). The ratio of fluorescence 665 over 620 was utilized as a readout of the assay.
8) Data analysis was performed using CBIS software (ChemInnovations, Inc).

Comment: Compounds that demonstrated an inhibition of >= 50% and 0.5 <= F_ratio <= 1.5 at 35 uM concentration are defined as actives in primary HTS assay. Compounds with Inhibition >= 50% that demonstrate F_ratios outside these boundaries are optically interfering with the assay (quenching or fluorescent) and the results are marked as "inconclusive".

The compounds identified as primary screening actives proceed to single-concentration confirmation stage. These compounds were retested in duplicate at a single 35 uM concentration in confirmation screen. Compounds with an average of >= 33% inhibition are considered active of the confirmation assay.

Substance SID26670119 was reordered for the confirmation assay. A different batch of the compound, SID7970403, was received and used.

Compounds with an IC50 < 100 uM are considered active in the dose response assay.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data-the score is correlated with % inhibition in the assay demonstrated by a compound at 35 uM concentration:
a. If primary % inhibition is less than 0%, then the assigned score is 0
b. If primary % inhibition is greater than 100%, then the assigned score is 40/(1+(F_ratio - 1)^2)
c. If primary % inhibition is between 0% and 100%, then the calculated score is (%inhibition)*0.4/(1+(F_ratio-1)^2)
d. For compounds retested at 35 uM, if primary % inhibition is greater than 100%, then the assigned score is 40
e. For compounds retested at 35 uM, if primary % inhibition is between 0% and 100%, then the calculated score is %inhibition)*0.4

2) Second tier (41-80 range) is reserved for dose-response confirmation data
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound's potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]

This empirical factor prorates the likelihood of target- or pathway-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the eIF4H assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account all the items discussed above is
Score = 44 + 6*(pIC50-3)*QC,
Where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay

External ID: JHICC_CaCC_Inh_Primary

Protocol: 1. Cell culture: Cells are routinely cultured in DMEM (high glucose, w glutamine), 10% FBS, 1%Pen/Strep.
2. Cell plating: Add 50 ul/well of 8,000/well re-suspended in media on BD PDK-pre-coated 384-well plate.
3. Incubate overnight at 37C and 5% CO2
4. Remove medium and add 25 ul /well of HBSS-HEPES (1x) (pH 7.4) to cells
5. Prepare 7.5X compound plates and control plates on Cybi-Well system: test compounds are prepared using assay buffer; controls are assay buffer(IC0), activator control ionomycin and inhibitor control NFA (all with DMSO concentrations matched to that of test compounds)
6. Add 5 microl drugs/compounds and controls to cell plates by Cybi and incubate at RT for 20 min.
7. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader
8. Prepare 8.5x trigger solution containing 4.25 uM ionomycin in 138mM iodide-HBSS solution (pH 7.4)
9. Measure fluorescence for 10 seconds at 1Hz to establish baseline
10. Add 5 ul of trigger solution into the cell plates on FDSS and continue measuring fluorescence for 50 seconds
11. Calculate ratio readout as F(max-min)/F0
12. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors [15].
13. Calculate B scores [16] for test compounds using ratios calculated in Step 11.
17. Outcome assignment: If the B score of the test compound is more than 3 times the standard deviation (SD) of the B scores of ratios of the library compounds (>=3*SD), AND the B score of initial fluorescence intensity is within 5 times the standard deviation of the B scores of the library compounds, the compound is designated in the Outcome as active as an inhibitor/blocker of the CaCC (TMEM16A) channels. Otherwise, it is designated as inactive.
18. Score assignment: An active test compound is assigned a score between 0 and 100 by calculation of with normalized ratioBScore, ratioBScore as in the result definition. Among the active compounds in the assay, the activity score range is 100-25. All inactive test compounds are assigned to the score 0.


List of reagents

(1)Cells (TMEM16A/YFP-H148Q/ I152L/HEK293) from JHICC.
(2)PBS: pH7.4 (Gibco, Cat#10010)
(3)Medium: DMEM (Cellgro, Cat# 10-013-CV )
(4)Fetal Bovine Serum (Gibco, Cat# 26140)
(5)200 mM L-Glutamine(Gibco, Cat#25030)
(6)Penicillin-Streptomycin(Mediatech, Cat#30-001-CI)
(7)Trypsin-EDTA: 0.25% Trypsin (Gibco, Cat#25300)
(8)G418 (100X): 50mg/mL(filtered) (Gibco, Cat#11811-031)
(9)HEPES (Sigma, Cat#H4034)
(10)BD Biocoat 384-well plates (BD, Cat#(35)4663 and Lot #7346273)
(11)Hygromycin (mediatech Cat# 30-240-CR)
(12)Niflumic acid (Sigma, Cat#:N0630)

Comment: Possible artifacts of this assay can include, but are not limited to: non-intended chemicals or dust in or on wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, and compounds that quench or emit light or fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.

External ID: JHICC_MrgX1_Agonist_Primary

Protocol: Protocol:
1) Seed cells (MRGX1-HEK293) 15,000/50ul/well on BD PDK-pre-coated 384-well plate, overnight.
2) Dump the medium thoroughly and add 20 microl of Fluo4 NW solution.
3) 37 degrees C and 5% CO2 incubation in the dark for 0.5 hr and Room temperature incubation in the dark for 0.5 hr.
4) Mount the cell plate to FDSS, with compound plate from plate loading (1 by 1, 4 microl, 6x this step, 10x final); 2nd plate on Stage II (10 plate/2nd addition plate, 6 microl, 5x this step, 6.67x final) and 3rd plate on Stage III (10 plate/3rd addition plate, 6 microl, 6x this step, 6x final).
5) Read for the first 2 additions for 10 plates (~5 min/plate; total 55 min)
6) After that, read the 3rd addition of all 10 plates (~3 min/plate)
7) Change tips and repeat step 5 to step 7.
8) Calculate ratio readout as F(max-min)/F0
9) Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors [6].
10) Calculate B scores [7] for test compounds using ratios calculated in Step 9.
11) Outcome assignment: If the B score of the test compound is more than 3 times the standard deviation (SD) of the B scores of ratios of the library compounds (>=3*SD), AND the B score of initial fluorescence intensity is within 5 times the standard deviation of the B scores of the library compounds, the compound is designated in the Outcome as active as an agonist hit of the MrgX1 target. Otherwise, it is designated as inactive.
12) Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of with normalized ratioBScore, ratioBScore as in the result definition. Among the active compounds in the assay, the activity score range is 100-5. All inactive test compounds are assigned to the score 0.

List of reagents
(1) Cells (MrgX1 stably expressed HEK293 cells) from Dr. Xinzhong Dong.
(2) PBS: pH7.4 (Gibco, Cat#10010)
(3) Medium: DMEM (Mediatech 10-014-CV)
(4) Fetal Bovine Serum (Gibco, Cat# 26140)
(5) 200 mM L-Glutamine(Gibco, Cat#25030)
(6) Penicillin-Streptomycin(Mediatech, Cat#30-001-CI)
(7) Trypsin-EDTA: 0.25% Trypsin (Gibco, Cat#25300)
(8) G418 (100X): 50mg/mL(filtered) (Gibco, Cat#11811-031)
(9) HEPES (Sigma, Cat#H4034)
(10) BD Biocoat 384-well plates (BD, Cat#(35)4663 and Lot #7346273)
(11) Fluo-4 NW Calcium Assay Kit (Invitrogen Cat# F36205)
(12) BAM8-22 (Tocris, Cat#: 1763)

Comment: Possible artifacts of this assay can include, but are not limited to: non-intended chemicals or dust in or on wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, and compounds that quench or emit light or fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.

External ID: SBCCG-A415-tim10-Primary-Antagonist-Assay

Protocol: Assay Materials:
Yeast Strain: ySHDSTim10 Isogeneic Control yeast strain (TIM10 strain)
Growth Media: YPD broth (TEKNOVA)
SD Assay Media: 1.2 g/L Yeast Nitrogen Base w/o amino acids, 5 g/L Ammonium Sulfate, 20 g/L Dextrose, 13.8 g/L Succinate, supplemented with 1 X Amino Acid Mix (2 g/L) (Sunrise Science Products, San Diego, CA)
SD Minimal Media: 1.2 g/L Yeast Nitrogen Base w/o amino acids, 5 g/L Ammonium Sulfate, 13.8 g/L Succinate, (Sunrise Science Products, San Diego, CA)
Assay Plate: Corning 1536 Well White Plate (Catalogue #: 3725)
Detection Reagent: Bactiter-GLO (Promega)

I. Compound Addition:

1. Using LabCyte Echo, transfer 20 nL from a 2 mM Echo qualified plate containing test compounds into assay plate columns 3 - 48 (final concentration of test compounds is 10 uM, 0.5 % DMSO). Transfer 20 nL of DMSO to positive and negative control wells in columns 1 - 4.
2. Centrifuge plates at 1000 rpm for 1 min.

II. Set up of tim10-1 assay:

The day before the screen, frozen culture was thawed at room temperature and resuspended in growth media at a cell density of 5x10^5/ml in approx. 100 ml. The culture was grown overnight at 25 oC with shaking (225 rpm).
3. In the morning of the Set-Up day prepare sufficient amount of SD Assay Media for negative control and compound wells and SD Minimal Media for positive control wells in order to obtain enough yeast cell culture for plating the desired number of 1536 well plates with 4 ul yeast cell suspension / well.
4. Count the yeast cells from the overnight Growth Media culture.
5. Dilute yeast to a final concentration of 2000 yeast/well in SD Media.
6. Pellet at 2400 rpm for 5 min at RT. Aspirate off supernatant.
7. Add 25 ml of sterile Water. Re-suspend cells by gently shaking. Pellet again at the same conditions, and wash the yeast cells in 25ml sterile water a second time.
8. Re-suspend in SD Assay Media for negative control and compound wells
9. For positive control wells, use SD Minimal Media with no yeast.
10. Add 4 ul yeast cells per well using combi and cover each plate with plastic lid.
11. Spin the plates at 2000 rpm for 1 min, incubate at 25 oC, inverted in a stack of 4, wrapped in saran wrap for 22-24 hours.

IV. Reading plates:
12. After 22-24 hours of incubation, add 3 ul of substrate solution (should be at RT) to all the wells of each plate using combi.
13. Plates are spun again at 2000 rpm, and left at RT for 15 min.
14. Read plates using a Perkin Elmer ViewLux using a luminescence protocol.

Comment: Compounds with %Activity >= 40% are defined as "active" in this assay.

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary and single-concentration confirmation screening data.
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30.
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.

2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay

External ID: BCCG-A237-eIF4H-FP-Assay

Protocol: HTS Assay Protocol

Assay materials:
1) His6-eIF4H (Obtained from Assay Provider)
2) 5'-fluorescein-labeled poly(U)10 (Purchased from Dharmacon)
3) Assay Buffer: 5 mM Hepes pH 7.5, 0.005 % Tween-20

Procedure:
1. Dispense 2 ul of assay buffer into columns 1 and 2 of a black, Corning (#2725) 1536 well assay plate.
2. Add 2 ul of eIF4H protein, 30 nM final concentration into columns 3-48.
3. Add 2 ul of 5'-fluorescein-labeled poly(A)10 2.5 nM final concentration into columns 1-48.
4. Using a HighRes biosolutions pintool dispense 70 nl of 2 mM compounds in DMSO to columns 5-48.
5. Using a HighRes biosolutions pintool dispense 70 nl of DMSO to columns 1-4.
6. Read plate on a BMG PHERAstar at 485/520/520nm in Fluorescence Polarization mode.
i. Positioning delay = 0.0
ii. Flashes/well = 10
7. Data analysis was performed using CBIS software (ChemInnovations, Inc).
8. Fluorescence intensity of each sample was normalized to the average fluorescence intensity value of the plate negative control wells to calculate F_ratio parameter.

Dose Response Protocol

Assay materials:
1) His6-eIF4H (Obtained from Assay Provider)
2) 5#-fluorescein-labeled poly(U)10 (Purchased from Dharmacon)
3) Assay Buffer: 5 mM Hepes pH 7.5, 0.005 % Tween-20

Procedure:
1. Using a Labcyte Echo, DMSO and test compounds are transferred to wells of a black, Corning 1536 well assay plate. DMSO only is transferred to columns 1-4 (Control wells), while varying volumes of test compounds are transferred to columns 5-48 to achieve the desired test concentrations. Compounds are transferred from a 2 mM stock to give the stated final concentration. Test compound wells in the assay plate are back-filled with DMSO to equalize final assay concentrations.
2. After compounds have been added, dispense 2 ul of assay buffer into columns 1 and 2 of a black, Corning (#2725) 1536 well assay plate.
3. Add 2 ul of eIF4H protein, 30 nM final concentration into columns 3-48.
4. Add 2 ul of 5#-fluorescein-labeled poly(A)10 2.5 nM final concentration into columns 1-48.
5. Read plate on a BMG PHERAstar at 485/520/520nm in Fluorescence Polarization mode.
i. Positioning delay = 0.0
ii. Flashes/well = 10
6. Data analysis was performed using CBIS software (ChemInnovations, Inc).
7. Fluorescence intensity of each sample was normalized to the average fluorescence intensity value of the plate negative control wells to calculate F_ratio parameter.

Comment: Compounds that demonstrated % activity of >= 50 % and 0.4 =< F_ratio <= 1.5 at 35 uM concentration are defined as actives of the primary screen in this assay. Compounds with activity >50% that demonstrate F_ratios outside these boundaries are optically compounds interfering with the assay (quenching or fluorescent) and the results are marked as "inconclusive".

Selected compounds from the primary screen were retested and those that confirmed in duplicate at 20uM concentration were considered "active". If the results of the duplicates were not consistent they are marked as "inconclusive."

Compounds were tested in a dose response assay and those with an IC50 < 100 uM are considered to be "active."

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data-the score is correlated with %Inhibition in the assay demonstrated by a compound at 35 uM concentration:
a. If primary %Inhibition is less than 0%, then the assigned score is 0
b. If primary %Inhibition is greater than 100%, then the assigned score is 40/(1+(F_ratio - 1)^2)
c. If primary %Inhibition is between 0% and 100%, then the calculated score is (%Inhibition)*0.4/(1+(F_ratio-1)^2)

2) Second tier (41-80 range) is reserved for dose-response confirmation data
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound's potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]

This empirical factor prorates the likelihood of target- or pathway-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the eIF4H assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account all the items discussed above is
Score = 44 + 6*(pIC50-3)*QC,
Where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in to this assay

External ID: PRPC_INH_TRFRET_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as inhibitors of PRPc cell surface expression. This assay detects PrPc on the surface of living LD9 fibroblast cells using two PRPC-specific antibodies which carry donor and acceptor fluorophores, respectively.

In this assay, the antibodies are directed against distinct, non-overlapping PrPc epitopes. Antibody occupancy of both epitopes of a PrPc molecule results in FRET signal emission. Free antibody or single antibody occupancy is not detected. The assay employs long half-life lanthanide donors (Europium or Terbium cryptate) and acceptor fluorophores XL665 or d2: SAF32 (aa53-93) and D18 (aa133-157) labeled with the donor and acceptor fluorophores, respectively. Ratios of 665 nm to 620 nm measurements are calculated, to take into account non-specific absorption of 620 nm light by the assay matrix. Compounds are tested in singlicate at a final nominal concentration of 13.8 microM.

Protocol Summary:

The LD9 cell line (subclone of L929, which is a mouse fibroblast cell line) was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of Opti-MEM I Reduced Serum Medium (no phenol red) supplemented with 5% v/v Bovine Growth Serum Supplemented Calf and 1X Penicillin-Streptomycin.

The day before the assay, 3 uL of cell growth media was dispensed into the first two columns of 1536 well microtiter plates and 1500 cells in 3 uL of cell growth media were seeded into the remaining wells. Next, 42 nL of test compound in DMSO, Brefeldin A (58 uM final concentration) in DMSO, or DMSO alone (1.38% final concentration) were dispensed to the appropriate wells. The plates were then incubated for 21 hours at 37 C, 5% CO2, and 95 % RH.

The assay was started by adding 1 uL of SAF32-Tb at a final concentration of 0.072ug/mL and 1ul of D18-d2 at a final concentration of 0.66ug/mL to all wells, respectively. Labeled Antibodies were prepared in 1X PBS. Plates were centrifuged and after 3 hours of incubation at room temperature, well TRFRET was read on a ViewLux microplate reader (Perkin Elmer, Turku, Finland). Measurements were performed by exciting the plates at 340 nM, and monitoring well fluorescence at 618 nm (Tb) and 671 nm (d2) with the ViewLux microplate reader.

To normalize data, values measured from both fluorescence emission wavelengths were used to calculate a ratio for each well, according to the following mathematical expression:

Ratio = I671nm / I618nm

Where:

I represents the measured fluorescence emission intensity at the enumerated wavelength in nm.

The percent inhibition was calculated from the median ratio as follows:

%_Inhibition = 100 * ( ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) ) )

Where:

Test_Compound is defined as wells containing cells, test compounds and DMSO.
High_Control is defined as wells containing cells, Brefeldin A and DMSO.
Low_Control is defined as the median of the wells containing test compounds.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-18, and for inactive compounds 18-0.

List of Reagents:

LD9 cells (supplied by Assay Provider)
Opti-MEM Reduced Serum Medium (Life Technologies, part 11058-021)
Bovine Growth Serum Supplemented Calf (Hyclone, part SH30541.03)
Penicillin-Streptomycin (100X) (Life Technologies, part 15140-122)
Brefeldin A (Control, Sigma, part B7651)
SAF32-Tb (Cisbio, custom labeled antibody)
D18-d2 (Cisbio, custom labeled antibody)
PBS (Life Technologies, part 10010-031)
Detachin Cell Detachment Reagent (Genlantis, part T100100)
T-175 Flasks (Nunc, part 159910)
1536-well plates (Corning, part 7298)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: MH083154

Protocol: Materials
MATERIALS
MEF-GFP-LC3 cells (provided by Dr. Xiao-Ming Yin, University of Pittsburgh)
384 well flat bottom microplates, collagen-coated (Falcon Biocoat)
DMEM growth medium (Gibco), supplemented with 10% Fetal bovine serum (HyClone) and antibiotics
Thapsigargin (Molecular Probes T-7459), dissolved in DMSO at 1 mM and frozen in aliquots at -20oC.
DMSO (Sigma 15,493-8)
Formaldehyde, 37 wt. % solution in water, A.C.S. reagent (Sigma 252549)
Test compounds plated into 384 well microplates (Greiner polypropylene).
Hanks balanced salt solution (HBSS, Fisher SH3026802)
Hoechst 33342 (Molecular Probes/Invitrogen H-1399)

PROTOCOL
Day 1. Cell seeding.
Plate 5,000 MEF-GFP-LC3 cells/40 [micro]L complete growth medium into the wells of collagen-coated 384 well microplates and incubate overnight at 37oC, 5% CO2.
Day 2. Compound treatment.
Thaw compound plates containing 2 [micro]L of 1 mM drug in DMSO.
Reconstitute wells A3:P22 of library compound plates in 38 [micro]L complete growth medium. Intermediate concentrations are 50 [micro]M compound and 5% DMSO
Load appropriate control wells on reconstituted library compound plates with 40 [micro]L of positive and negative controls (5% DMSO) and 5 [micro]M thapsigargin in 5% DMSO (Biomek2000, Perkin Elmer Multiprobe).
Transfer 10 [micro]L of treatment solution from reconstituted library compound plates to assay plates. (Perkin Elmer Janus MDT) and incubate for 18h at 37oC, 5% CO2. The final assay concentration will be 10 [micro]M compound and 1% DMSO.

Day 3. Fixation, staining, and analysis.
Prepare fixative (10.7% formaldehyde and 26.7 [micro]g/ml Hoechst 33342 in HBSS).
Dispense 30 [micro]L of fixative directly into all plates (Titertek MAP-C2).
Incubate for 30 min at room temp. under dimmed light.
Aspirate off fixative and wash plates three times with 50 [micro]L PBS (MAP-C2).
Seal plates and analyze on the ArrayScan Vti (Thermo Fisher Cellomics) for autophagosomal GFP-LC3 accumulation.
Acquire images in two channels (DAPI/FITC) using a 10x objective and an XF100 filter set on the ArrayScan VTi.

ANALYSIS
Analyze images by the Compartmental Analysis Bioapplication, configured to detect Hoechst 33342 stained nuclei, cytosolic GFP expression, and autophagosomes as GFP-expressing punctate objects located in the cytosol. First, nuclear and cytosolic areas were separated to avoid nuclear punctate objects to be included. Punctate structures were then detected in the cytosol by thresholding over local background. A selection threshold was then set using DMSO-treated cells to calculate the percentage of cells positive for autophagosome formation.

Comment: HCS parameters reported
1. %HIGH_RingSpotCountCh2: The percentage of cells containing GFP spots (Channel 2). The percentage of responders is computed on a plate by plate basis.
2. Z-score_%HIGH_RingSpotCountCh2: Z-score of the percentage of cells containing GFP spots (Channel 2).
3. HCS_cmpd_conc: Primary HCS compound concentration in [micro]M
4. MEAN_RingAvgIntenCh2. The average GFP intensity in the cytosol (Channel 2). The average intensity is reported on a per cell basis.
5. MEAN_RingSpotTotalAreaCh2. The total (integrated) intensity of GFP spots per cell (Channel 2). The total spot intensity is reported on a per cell basis.
6. SelectedObjectCountPerValidField. The number of nuclei per imaging field.
7. % Toxicity. % toxicity = 1-(SelectedObjectCountPerValidField in sample well / average SelectedObjectCountPerValidField of all MIN controls on the plate)*100
8. Assay Date : Date the HTS assay was performed

The high-content cell-based screen for modulators of autophagy conducted by the PMLSC utilized a Z-score statistical scoring method to identify active compounds (Brideau et al., 2003)
Brideau, C., Gunter, B., Pikounis, B., and Liaw, A. (2003). Improved statistical methods for hit selection in high-throughput screening. Journal of Biomolecular Screening 8, 634-647. 14711389

Target activity score (Z-score_%HIGH_RingSpotCountCh2)
The Z-score for a compound was computed on a plate by plate basis. The Z-score for the percentage of cells containing autophagosomes in Channel 2 (Xi) was defined as Xi = (Xi - Xm)/Sm, where Xm is the mean of all the percentage of cells containing autophagosomes values in wells A3:P22 on the microplate, and Sm is the standard deviation of these values. A cut-off Z-score of greater than 3 was selected as the definition as the active criterion.

Fluorescent outliers. (MEAN_RingAvgIntenCh2)
The average GFP intensity per cell was calculated on a per cell basis. A cut-off score of 1500 was selected as the definition for a fluorescence outlier.

Cytotoxicity outliers (% toxicity)
% toxicity was computed on a plate by plate basis. % toxicity was defined as 1-(cell density in sample well / average cell density of all MIN controls on the plate)*100. A cut-off score of more than 80% was selected as the definition for a cytotoxic compound.


Definition of an active compound
Z-score_%HIGH_RingSpotCountCh2 > 3, MEAN_RingAvgIntenCh2 > 1500, and % toxicity < 80%

PUBCHEM_ACTIVITY_OUTCOME
1 inactive. Substance is considered inactive when the Z-score_%HIGH_RingSpotCountCh2 > 3 at 10 [micro]M.
2 active. Substance is considered inactive when the Z-score_%HIGH_RingSpotCountCh2 > 3, MEAN_RingAvgIntenCh2 < 1500, and % toxicity < 80% at 10 [micro]M

PUBCHEM_ACTIVITY_SCORE
0-40 range is reserved for primary HTS data
a)If the substance is considered active the score is 40
b)If the substance is inactive the score is 0

Secondary assay testing paradigm:
Confirmation of inhibition of autophagosome formation in 2 independent tests at 10 [micro]M.
Concentration Response IC50 < 10 [micro]M in the autophagy inducer HCS assay.
Structural Verification
Indications of Specificity & Selectivity ~ PubChem X-target Query
Evidence of SAR.

Confirmed concentration dependent actives would be tested for A) autophagosome formation in a different cell line stably expressing LC3-GFP and B) for long lived protein degradation.

External ID: CHOK1_ANT_FLUO8_1536_1X%INH CSRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that non-selectively inhibit Gq signaling. In this assay, the parental CHO cell line (not transfected with any GPCRs) is used to monitor inhibition of Gq activity by test compound. Cells are incubated with test compounds, followed by measurement of intracellular calcium as monitored by the FLUO-8 fluorescent, cell permeable calcium indicator dye. As designed, compounds that act as Gq antagonists will decrease calcium mobilization, resulting in decreased relative fluorescence of the indicator dye, and thus decreased well fluorescence. These compounds are considered nonselective Gq inhibitors. Compounds are tested in singlicate at a nominal concentration of 5.6 uM.

Protocol Summary:

The CHO cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of Ham's F-12 Nutrient Media (F-12) supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 25 mM HEPES, and 1X antibiotic mix (penicillin, streptomycin, and neomycin).

The day before the assay, 2000 cells in 3 uL of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 C, 5% CO2, and 95 % RH for 23 hours. Next, 2 uL of the fluorogenic Fluo-8 intracellular calcium indicator mixture with 1 mM trypan red plus (prepared according to the manufacturer's protocol) was added to each well. After incubation for 1 hour at 37 C, 5% CO2, and 95 % RH, 25 nL of test compound in DMSO, or DMSO alone were dispensed to the appropriate wells. The assay was started after an additional 30 minute incubation at room temperature, by performing a basal read of plate fluorescence (470-495 nm excitation and 515-575 nm emission) for 6 seconds on the FLIPR Tetra (Molecular Devices). Next, 15 nL of ATP in DMSO (EC84 average response), or DMSO alone were dispensed to the appropriate wells. Then a real time fluorescence measurement was immediately performed for the remaining 94 seconds of the assay.

A ratio for each well was calculated to normalize assay data, according to the following mathematical expression:

Ratio = I_Max / I_Min

Where:

I_Max represents the maximum measured fluorescence emission intensity over the 100 second read.
I_Min represents the minimum (basal) measured fluorescence emission intensity before compound was added.

Percent inhibition was calculated from the median ratio as follows:

% Inhibition = ( 1 - ( ( Ratio_Test_Compound - Median_Ratio_High_Control ) / ( Median_Ratio_Low_Control - Median_Ratio_High_Control ) ) ) * 100

Where:

Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing ATP Challenge.
High_Control is defined as wells containing DMSO alone (no ATP).

A mathematical algorithm was used to determine nominally inhibiting compounds in the screen. Two values were calculated for each assay plate: (1) the average percent inhibition of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % inhibition than that particular plate's cutoff parameter was declared active.

PubChem Activity Outcome and Score:

The PubChem Activity Score range for active compounds is 100-22, and for inactive compounds 61-0.

List of Reagents:

CHO cells (ATCC, part CCL-61)
Fluo-8 No Wash Calcium Assay Kit (AAT Bioquest, part 36316)
Trypan red plus (ABD Bioquest, part 2456)
Ham's F-12 media (Invitrogen, part 11765-054)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Fetal Bovine Serum (Invitrogen, part 16140-071)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
T-175 tissue culture flasks (Nunc, part 159910)
Agonist: ATP (Sigma-Aldrich, part A6559)
1536-well plates (Greiner, part 789072)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that quench or emit fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.

External ID: DAX1-FULL_INH_LUMI_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that can reduce DAX-1 activity. DAX-1 has been shown to inhibit SF-1-mediated transactivation (5).

SF-1 (steroidogenic factor 1; NR5A1) has been shown to function as a transcription factor for a variety of different steroidogenic enzyme genes in the adrenal gland and gonads. Specifically, SF-1 has been shown to induce differentiation of mesenchymal stem cells into steroidogenic cells with concomitant strong induction of StAR expression.

This assay employs HEK293T cells co-transfected with plasmids expressing SF-1 and a luciferase reporter under control of the StAR promoter together with an expression plasmids for DAX-1, followed by treatment with test compounds. Under normal conditions, DAX-1 represses the SF-1-induced expression of the luciferase reporter. As designed, a compound that inhibits DAX-1 activity will reduce DAX1-mediated repression of SF-1, thereby allowing SF-1 to increase StAR promoter activity, resulting in increased well luminescence. Compounds will be tested in singlicate at a nominal concentration of 6.8 uM.

Protocol Summary:

HEK293 cells were routinely cultured in T-175 flasks containing 25 mL of DMEM media supplemented with 10% v/v fetal bovine serum and 1% v/v antibiotic-antimycotic mix at 37 C, 5% CO2 and 95% relative humidity (RH). The day prior to run the assay, the HEK293 cells were harvested using 5 mL of TrypLE reagents and seeded in fresh media at a density of 10 million cells per T175 flask. The following day, cells were transfected with 5 mL of serum-free OptiMEM containing 28 ug of the StAR promoter luciferase reporter plasmid, 14 ug of the SF-1 expressing palsmid, 21 ug of the DAX-1 expressing plasmid and 100 uL of transfection reagent. Four hours post transfection, cells were harvested using 5 mL of preheated TrypLE and resuspended at a concentration of 800,000 cells per mL in phenol-red free DMEM supplemented as above. In the absence of a pharmacological positive control, DAX-1 inhibition was mimicked by transfecting cells with an empty vector in place of the DAX-1 expressing vector.

The assay was started by dispensing 5 uL of cell suspension into each well of white, solid-bottom 1536-well plates using a flying reagent dispenser (Aurora). The first three columns received control cells (no DAX-1 expressed) whereas the rest of the plate was dispensed with DAX1-transfected cells. The plates were then treated with 34 nL/well of test compounds or DMSO (final concentration 0.68%) on DAX-1 cells and Control cells using a PinTool transfer unit (GNF). Plates were incubated for eighteen hours at 37 C, 5% CO2 and 95%RH. Plates were then removed from the incubator and equilibrated to room temperature for 10 minutes. Luciferase activity was detected by addition of 5 uL of SteadyLite reagent to each well. After a 15 minute incubation time, light emission was measured with the ViewLux reader (PerkinElmer).

The percent inhibition of each test compound was calculated as follows:

%_Inhibition = ( 1 - ( median_positive_control - test_compound ) / (median_positive_control - median_negative_control ) * 100

Where:

Positive control is defined as wells containing control cells (no DAX-1 expressed) treated with DMSO.
Negative control is defined as wells containing DAX-1 cells treated with DMSO.
Test compound is defined as wells containing DAX-1 cells containing test compound.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally active compounds. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-4, and for inactive compounds 4-0.

List of Reagents:

pGL2_1.3 kb_StAR luciferase reporter plasmid (Assay Provider)
pSG.SF-1 plasmid (Assay Provider)
pSV.DAX-1 plasmid (Assay Provider)
pSG5 empty vector (Assay Provider)
HEK293 cells (ATCC, part CRL-1573)
DMEM (Invitrogen, part 11965)
FBS (Hyclone, part SH30088.03)
Antibiotic-Antimycotic 100X Liquid Solution (Gibco, part 15240)
TransIT 293 (Mirus Corporation, part MIR-2700)
OptiMEM (Invitrogen, part 31985)
TrypLE Trypsin Replacement Enzyme (Invitrogen, part 12604)
SteadyLite Reagent (PerkinElmer, part 6016989)
1536-well plates (Greiner part 789173)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well luminescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: MH081226: Primary HTS for Inhibitors of a Novel Necrotic Cell Death Pathway # Jurkat FADD-/- Model.

Protocol: Necroptosis Assay Protocol TNF-alpha Induced Cell Death in Jurkat FADD-/- Cells.

Based on the assay development experiments performed by the PMLSC the following assay conditions were selected for the 384-well TNF-alpha Jurkat FADD-/- necroptosis screen:
1. 20,000 Jurkat FADD-/- cells/well were seeded in 40 uL of complete media in white opaque 384-well microtiter plates.
2. Plate controls, compounds (10 uM final in well) and 40 ng/mL TNF-alpha were added in a single addition and cells were incubated for an additional 24 hrs at 37#C and 5% CO2.
3. 25 uL of Cell Titer Glo reagent was added to each well and the resulting luminescence measurement of cellular ATP levels was read on the Envision after 15 minutes at ambient temperature.

Materials:
1. Complete Media: RPMI1640 with L-glutamine, Penicillin-Streptomycin, 10% Newborn calf serum
2. Jurkat FADD-/- Cells: 20,000 cells/well in 40 μL complete media
3. TNF-alpha: 50 ug/mL stock in DMSO; final concentration = 40 ng/mL
4. Cell Titer Glo: use at 25 μL/well

Protocol:
1. Jurkat FADD-/- cells are seeded at 20,000 cells/well in 40 uL of complete media into 384-well white opaque-bottom polypropylene plates (Greiner Bio-one Cat #781080).
2.Compounds (10 uM final concentration) are added to the respective wells in a total volume of 10 uL/well.
3. Maximum and minimum controls (DMSO at 0.18% final concentration and TNF-alpha at 40 ng/mL final concentration respectively) are added to assay plates at 20 uL/well
4. TNF-alpha (40 ng/mL final concentration) is added at 10 uL/well to respective wells with compounds.
5. Cell plates are incubated overnight at 37#C and 5% CO2.
6. 25 uL Cell Titer Glo is added to each well and the luminescence signal is read on the Envision after 15 minutes.

Plate Controls:
Max: 0.18% DMSO in complete media
Min: 40 ng/mL TNF-alpha0.08% DMSO) + 0.1% DMSO in complete media (final DMSO concentration = 0.18%)

Comment: The TNF-alpha induced Jurkat FADD-/- necroptosis Assay HTS run at the PMLSC utilized % activation calculated from maximum (n=32) and minimum (n=24) plate controls, with a hit criteria of >/= 50% activation to identify active compounds.

TNF-alpha Jurkat FADD-/- necroptosis Assay Activity scoring rules:

PUBCHEM_ACTIVITY_OUTCOME

1 - Substance is considered inactive when the % activation is < 50%
2 - Substance is considered active when % activation is >/= 50%
3 - Substance activity outcome is inconclusive

PUBCHEM_ACTIVITY_SCORE

0-40 scoring range is reserved for primary HTS data
a) if the % activation is >/= 50%, the score is 40.
b) if the % activation is < 50 %, the score is 0.

Definition of a Hit:
Rapid HTS screen ≥ 50% inhibition of death at 10 uM.
Confirmation of ≥ 50% inhibition of TNF-alpha induced Jurkat FADD-/- cell death in 2 independent tests.
Concentration Response AC50 < 20 uM in the Jurkat FADD-/- cell death model.
Structural Verification
Indications of Specificity & Selectivity ~ PubChem X-target Query
Evidence of SAR.